Pharmacologically induced transgene ablation system

ABSTRACT

The present invention relates to gene therapy systems designed for the delivery of a therapeutic product to a subject using replication-defective virus composition(s) engineered with a built-in safety mechanism for ablating the therapeutic gene product, either permanently or temporarily, in response to a pharmacological agent—preferably an oral formulation, e.g., a pill. The invention is based, in part, on the applicants&#39; development of an integrated approach, referred to herein as “PITA” (Pharmacologically Induced Transgene Ablation), for ablating a transgene or negatively regulating transgene expression. In this approach, replication-deficient viruses are used to deliver a transgene encoding a therapeutic product (an RNA or a protein) so that it is expressed in the subject, but can be reversibly or irreversibly turned off by administering the pharmacological agent; e.g., by administration of a small molecule that induces expression of an ablator specific for the transgene or its RNA transcript.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/247,306, filed Sep. 28, 2011, which is a continuation-in-part ofInternational Patent Application No. PCT/US2011/030213, filed Mar. 28,2011, which claims the benefit under 35 USC 119(e) of U.S. ProvisionalPatent Application No. 61/318,752, filed Mar. 29, 2010, all of which areincorporated herein by reference.

2. BACKGROUND OF THE INVENTION

Gene therapy involves the introduction of genetic material into hostcells with the goal of treating or curing disease. Many diseases arecaused by “defective” genes that result in a deficiency in an essentialprotein. One approach for correcting faulty gene expression is to inserta normal gene (transgene) into a nonspecific location within the genometo replace a nonfunctional, or “defective,” disease-causing gene. Genetherapy can also be used as a platform for the delivery of a therapeuticprotein or RNA to treat various diseases so that the therapeutic productis expressed for a prolonged period of time, eliminating the need forrepeat dosing. A carrier molecule called a vector must be used todeliver a transgene to the patient's target cells, the most commonvector being a virus that has been genetically altered to carry normalhuman genes. Viruses have evolved a way of encapsulating and deliveringtheir genes to human cells in a pathogenic manner and thus, virusgenomes can be manipulated to insert therapeutic genes.

Stable transgene expression can be achieved following in vivo deliveryof vectors based on adenoviruses or adeno-associated viruses (AAVs) intonon dividing cells, and also by transplantation of stem cells transducedex vivo with integrating and non-integrating vectors, such as thosebased on retroviruses and lentiviruses. AAV vectors are used for genetherapy because, among other reasons, AAV is nonpathogenic, it does notelicit a deleterious immune response, and AAV transgene expressionfrequently persists for years or the lifetime of the animal model (seeShyam et al., Clin. Microbiol. Rev. 24(4):583-593). AAV is a small,nonenveloped human parvovirus that packages a linear strand of singlestranded DNA genome that is 4.7 kb. Productive infection by AAV occursonly in the presence of a helper virus, either adenovirus or herpesvirus. In the absence of a helper virus, AAV integrates into a specificpoint of the host genome (19q 13-qter) at a high frequency, making AAVthe only mammalian DNA virus known to be capable of site-specificintegration. See, Kotin et at., 1990, PNAS, 87: 2211-2215. However,recombinant AAV, which does not contain any viral genes and only atherapeutic gene, does not integrate into the genome. Instead therecombinant viral genome fuses at its ends via inverted terminal repeatsto form circular, episomal forms which are predicted to be the primarycause of the long term gene expression (see Shyam et at., Clin.Microbiol. Rev. 24(4):583-593).

Virtually all pre-clinical and clinical applications of gene therapyhave used vectors that express the transgene from a constitutivepromoter, which means it is active at a fixed level for as long as thevector genome persists. However, many diseases that are amenable to genetherapy may need to have expression of the transgene regulated. Severalsystems have been described which that are based on the generalprinciple of placing a gene of interest under the control of adrug-inducible engineered transcription factor in order to positivelyinduce gene expression (Clackson et at., 1997, Curr Opin Chem BioI, 1(2): 210-8; Rossi et at., Curr Opin Biotechnol, 1998.9(5): p. 451-6).The various systems can be divided into two classes. In the first, aDNA-binding domain that is allosterically regulated by inducers such astetracyclines, antiprogestins, or ecdysteroids is coupled to atransactivation domain. The addition (or in some cases removal) of thedrug leads to DNA binding and hence transcriptional activation. In thesecond, allosteric control is replaced with the more general mechanismof induced proximity DNA binding and activation domains are expressed asseparate polypeptides that are reconstituted into an activetranscription factor by addition of a bivalent small molecule, referredto as a chemical inducer of dimerization or “dimerizer.” While thesesystems are useful in gene therapy systems that require inducingtransgene expression, they have not addressed the need to be able toturn off or permanently ablate transgene expression if it is no longerneeded or if toxicity due to long-term drug administration ensues.

3. SUMMARY OF THE INVENTION

The present invention relates to gene therapy systems designed for thedelivery of a therapeutic product to a subject usingreplication-defective virus composition(s) engineered with a built-insafety mechanism for ablating the therapeutic gene product, eitherpermanently or temporarily, in response to a pharmacologicalagent—preferably an oral formulation, e.g., a pill.

The invention is based, in part, on the applicants' development of anintegrated approach, referred to herein as “PITA” (PharmacologicallyInduced Transgene Ablation), for ablating a transgene or negativelyregulating transgene expression. In this approach, replication-deficientviruses are used to deliver a transgene encoding a therapeutic product(an RNA or a protein) so that it is expressed in the subject, but can bereversibly or irreversibly turned off by administering thepharmacological agent.

The invention presents many advantages over systems in which expressionof the transgene is positively regulated by a pharmacological agent. Insuch cases, the recipient must take a pharmaceutic for the duration ofthe time he/she needs the transgene expressed—a duration that may bevery long and may be associated with its own toxicity.

In one aspect, the invention provides a composition for AAV-mediateddelivery of a therapeutic product having a controlled transgeneexpression ablation system, said composition comprising (a) an AAVvector containing a nucleic acid molecule comprising: (i) a nucleic acidsequence encoding a therapeutic product operably linked to a promoterthat controls transcription; and (ii) at least one endonuclease ablationsite which comprises a sequence of at least 30 nucleic acid base pairswhich are specifically and selectively recognized by at least ten (10×)zinc fingers, said at least one endonuclease ablation site being locatedat least 5′ to the sequence encoding the therapeutic product; and (b) atleast one ablator which comprises a chimeric endonuclease comprising atleast ten copies of a zinc finger domain linked to a functionalendonuclease catalytic domain in operative association with a promoter,wherein transcription and/or ablation activity is induced in response toa pharmacological agent, said at least ten (10×) zinc finger domainspecifically and selectively recognizing said at least about 30 basepair sequence in said at least one endonuclease ablation site andcomprising at least 10 independently selected recognition helices. Inone embodiment, the endonuclease catalytic domain is a FokI catalyticdomain.

In a further embodiment, the nucleic acid molecule consists of adouble-stranded DNA molecule, wherein the at least one endonucleaseablation site is on a first strand of the DNA molecule and at least asecond endonuclease ablation is located on the second strand of the DNAmolecule, wherein said second endonuclease ablation site is distinctfrom said endonuclease ablation said on the first strand and isspecifically and selectively recognized by a different zinc finger.

In a further aspect, the ablator (b) is controlled by a cassette that isactivated by a transcription factor following being dimerized by apharmacologic agent, said cassette comprising two transcription units,wherein: (c) one of said two transcription units encoding the DNAbinding domain of the transcription factor fused to a binding domain forthe pharmacological agent in operative association with a firstpromoter; and (d) a second of said two transcription units encoding theactivation domain of the transcription factor fused to a binding domainfor the pharmacological agent in operative association with a secondpromoter.

In one embodiment, the first and second promoters are both constitutivepromoters and the pharmacological agent is a dimerizer that dimerizesthe domains of the transcription factor.

In another embodiment, the transcription of the promoter is controlledby a rapamycin—regulatable system and the pharmacological agent israpamycin or a rapalog.

In yet another embodiment, the unique nucleic acid sequence of leastabout a 30 base pair of (a)(ii) consists of contiguous nucleic acidswhich are less than 70% identical with any subsequence in the humangenome and no more than 8 contiguous identical positions with anysub-sequence in the human genome.

Other aspects and advantages of the invention will be readily apparentfrom the following Detailed Description of the Invention.

As used herein, the following terms will have the indicated meaning:

“Unit” refers to a transcription unit.

“Transgene unit” refers to a DNA that comprises (1) a DNA sequence thatencodes a transgene; (2) an ablation recognition site (ARS) containedwithin or flanking the transgene; and (3) a promoter sequence thatregulates expression of the transgene.

“Ablation recognition site” or “ARS” refers to a DNA sequence that (1)can be recognized by the ablator that ablates or excises the transgenefrom the transgene unit; or (2) encodes an ablation recognition RNAsequence (ARRS)

“Ablation recognition RNA sequence” or “ARRS” refers to an RNA sequencethat is recognized by the ablator that ablates the transcription productof the transgene or translation of its mRNA.

“Ablator” refers to any gene product, e.g., translational ortranscriptional product, that specifically recognizes/binds to either(a) the ARS of the transgene unit and cleaves or excises the transgene;or (b) the ARRS of the transcribed transgene unit and cleaves orprevents translation of the mRNA transcript.

“Ablation unit” refers to a DNA that comprises (1) a DNA sequence thatencodes an Ablator; and (2) a promoter sequence that controls expressionof said Ablator.

“Dimerizable transcription factor (TF) domain unit” refers to (1) a DNAsequence that encodes the DNA binding domain of a TF fused to thedimerizer binding domain (DNA binding domain fusion protein) controlledby a promoter; and (2) a DNA sequence that encodes the activation domainof a TF fused to the dimerizer binding domain (activation domain fusionprotein) controlled by a promoter. In one embodiment, each unit of thedimerizable domain is controlled by a constitutive promoter and the unitis utilized for control of the promoter for the ablator. Alternatively,one or more of the promoters may be an inducible promoter.

A “Dimerizable fusion protein unit” refers to (1) a first DNA sequencethat encodes a unit, subunit or fragment of a protein or enzyme (e.g.,an ablator) fused to a dimerizer binding domain and (2) a second DNAsequence that encodes a unit, subunit or fragment of a protein orenzyme, which when expressed and if required, activated, combine to forma fusion protein. This “Dimerizable fusion protein unit” may be utilizedfor a variety of purposes, including to activate a promoter for theablator, to provide DNA specificity, to activate a chimeric ablator bybringing together the binding domain and the catalytic domain, or toproduce a desired transgene. These units (1) and (2) may be in a singleopen reading frame separated by a suitable linker (e.g., an IRES or 2Aself-cleaving protein) under the control of single promoter, or may bein separate open reading frames under the control of independentpromoters. From the following detailed description, it will be apparentthat a variety of combinations of constitutive or inducible promotersmay be utilized in the two components of this unit, depending upon theuse to which this fusion protein unit is put (e.g., for expression of anablator). In one embodiment, the dimerizable fusion protein unitcontains DNA binding domains which include, e.g., zinc finger motifs,homeo domain motifs, HMG-box domains, STAT proteins, B3,helix-loop-helix, winged helix-turn-helix, leucine zipper,helix-turn-helix, winged helix, POU domains, DNA binding domains ofrepressors, DNA binding domains of oncogenes and naturally occurringsequence-specific DNA binding proteins that recognize >6 base pairs.

“Dimerizer” refers to a compound or other moiety that can bindheterodimerizable binding domains of the TF domain fusion proteins ordimerizable fusion proteins and induce dimerization or oligomerizationof the fusion proteins. Typically, the dimerizer is delivered to asubject as a pharmaceutical composition.

“Side effect” refers to an undesirable secondary effect which occurs ina patient in addition to the desired therapeutic effect of a transgeneproduct that was delivered to a patient via administration of areplication-defective virus composition of the invention.

“Replication-defective virus” or “viral vector” refers to a synthetic orartificial genome containing a gene of interest packaged inreplication-deficient virus particles; i.e., particles that can infecttarget cells but cannot generate progeny virions. The artificial genomeof the viral vector does not include genes encoding the enzymes requiredto replicate (the genome can be engineered to be “gutless”—containingonly the transgene of interest flanked by the signals required foramplification and packaging of the artificial genome). Therefore, it isdeemed safe for use in gene therapy since replication and infection byprogeny virions cannot occur except in the presence of the viral enzymerequired for replication.

“Virus stocks” or “stocks of replication-defective virus” refers toviral vectors that package the same artificial/synthetic genome (inother words, a homogeneous or clonal population).

A “chimeric engineered ablator” or a “chimeric enzyme” is provided whena sequence encoding a catalytic domain of an endonuclease ablator fusedto a binding domain and a sequence encoding a DNA binding domain of theendonuclease fused to a binding domain are co-expressed. The chimericengineered enzyme is a dimer, the DNA binding domains may be selectedfrom among, for example, zinc finger and other homeodomain motifs,HMG-box domains, STAT proteins, B3, helix-loop-helix, wingedhelix-turn-helix, leucine zipper, helix-turn-helix, winged helix, POUdomains, DNA binding domains of repressors, DNA binding domains ofoncogenes and naturally occurring sequence-specific DNA binding proteinsthat recognize >6 base pairs. [U.S. Pat. No. 5,436,150, issued Jul. 25,1995]. When a heterodimer is formed, the binding domains are specificfor a pharmacologic agent that induces dimerization in order to providethe desired enzymatic bioactivity, DNA binding specificity, and/ortranscription of the ablator. Typically, an enzyme is selected which hasdual domains, i.e., a catalytic domain and a DNA binding domain whichare readily separable. In one embodiment, a type II restrictionendonuclease is selected. In one embodiment, a chimeric endonuclease isdesigned based on an endonuclease having two functional domains, whichare independent of ATP hydrolysis. Useful nucleases include type II Sendonucleases such as FokI, or an endonuclease such as Nae I. Anothersuitable endonuclease may be selected from among intron endonucleases,such as e.g., I-TevI. Still other suitable nucleases include, e.g.,integrases (catalyze integration), serine recombinases (catalyzerecombination), tyrosine recombinases, invertases (e.g. Gin) (catalyzeinversion), resolvases, (e.g., Tn3), and nucleases that catalyzetranslocation, resolution, insertion, deletion, degradation or exchange.However, other suitable nucleases may be selected.

4. BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B. PITA DNA construct containing a dimerizabletranscription factor domain unit and an ablation unit. FIG. 1A is a mapof the following DNA construct, which comprises a dimerizabletranscription factor domain unit and an ablation unit:pAAV.CMV.TF.FRB-IRES-1xFKBP.Cre. FIG. 1B is a cartoon of thetranscription unit inserted into the plasmid backbone. A description ofthe various vector domains can be found in Section 8.1 herein.

FIGS. 2A and 2B. PITA DNA construct containing a dimerizabletranscription factor domain unit and an ablation unit. FIG. 2A is a mapof the following DNA construct, which comprises a dimerizabletranscription factor domain unit and an ablation unit:pAAV.CMV.TF.FRB-T2A-2xFKBP.Cre. FIG. 2B is a cartoon of thetranscription unit inserted into the plasmid backbone. A description ofthe various vector domains can be found in Section 8.1 herein.

FIGS. 3A and 3B. PITA DNA construct containing a dimerizabletranscription factor domain unit and an ablation unit. FIG. 3A is map ofthe following DNA construct, which comprises a dimerizable transcriptionfactor domain unit and an ablation unit:pAAV.CMV173.TF.FRB-T2A-3xFKBP.Cre. FIG. 3B is a cartoon of thetranscription unit inserted into the plasmid backbone. A description ofthe various vector domains can be found in Section 8.1 herein.

FIGS. 4A and 4B. PITA DNA construct containing a dimerizabletranscription factor domain unit and an ablation unit. FIG. 4A is a mapof the following DNA construct, which comprises a dimerizabletranscription factor domain unit and an ablation unit:pAAV.CMV.TF.FRB-T2A-2xFKBP.ISce-I. FIG. 4B is a cartoon of thetranscription unit inserted into the plasmid backbone. A description ofthe various vector domains can be found in Section 8.1 herein.

FIGS. 5A and 5B. PITA DNA construct containing a transgene unit. FIG. 5Ais a map of the following DNA construct, which comprises a transgeneunit: pENN.CMV.PL1oxP.Luc.SV40. FIG. 5B is a cartoon of thetranscription unit inserted into the plasmid backbone. A description ofthe various vector domains can be found in Section 8.2 herein.

FIGS. 6A and 6B. PITA DNA construct containing a transgene unit. FIG. 6Ais a map of the following DNA construct, which comprises a transgeneunit: pENN.CMV.PISceI.UC.SV40. FIG. 6B is a cartoon of the transcriptionunit inserted into the plasmid backbone. A description of the variousvector domains can be found in Section 8.2 herein.

FIG. 7. PITA DNA construct containing a dimerizable transcription factordomain unit and a transgene unit. FIG. 7 is a map of a vector thatcontains a transgene unit and a dimerizable transcription factor domainunit. A description of the various vector domains can be found inSections 8.1 and 8.2 herein.

FIGS. 8A-B. In vitro induction of luciferase after rapamycin treatment.FIG. 8A is a bar graph showing relative luciferase activity in cellsthat were transfected with the indicated DNA constructs (DNA constructs1 to 6) 48 hours after either being treated or not treated withrapamycin. FIG. 8B is a bar graph showing relative luciferase activityin cells that were transfected with the indicated DNA constructs (DNAconstructs 1 to 6) 72 hours after either being treated or not treatedwith rapamycin.

FIGS. 9A-D. In the in vivo model for a dimerizer-inducible system, fourgroups of mice received IV injection of AAV vectors containing thefollowing DNA constructs. FIG. 9A is a diagram of a DNA constructencoding GFP-Luciferase under the control of ubiquitous constitutive CMVpromoter, which was delivered to Group 1 mice via AAV vectors. FIG. 9Bis a diagram of DNA constructs encoding (1) a dimerizable transcriptionfactor domain unit (FRB fused with p65 activation domain and DNA bindingdomain ZFHD fused with 3 copies of FKBP) driven by the CMV promoter; and(2) AAV vector expressing GFP-Luciferase driven by a promoter induced bythe dimerized TF, which were delivered to Group 2 mice via AAV vectors.FIG. 9C is a diagram of a DNA construct encoding GFP-Luciferase underthe control of a liver constitutive promoter, TBG, which was deliveredto Group 3 mice via AAV vectors. FIG. 9D is a diagram of DNA constructsencoding (1) AAV vector expressing a dimerizable transcription factordomain unit driven by the TBG promoter; and (2) AAV vector expressingGFP-Luciferase driven by a promoter induced by the dimerized TF, whichwere delivered to Group 4 mice via AAV vectors.

FIGS. 10A-D. Image of 4 groups of mice that received 3×10¹¹ particles ofAAV virus containing various DNA constructs 30 minutes after injectionof luciferin, the substrate for luciferase. FIG. 10A shows luciferaseexpression in various tissues, predominantly in lungs, liver and muscle,in Group 1 mice before (“Pre”) and after (“Post”) rapamycinadministration. FIG. 10B shows luciferase expression, predominantly inliver and muscle in Group 2 mice before (“Pre”) and after (“Post”)rapamycin administration. FIG. 10C shows luciferase expressionpredominantly in liver and muscle after (“Post”) rapamycinadministration, and shows that there is no luciferase expression before(“Pre”) rapamycin administration in Group 3 mice. FIG. 10D showsluciferase expression is restricted to the liver (“Post”) rapamycinadministration and shows that there is no luciferase expression before(“Pre”) rapamycin administration.

FIGS. 11A-D. Image of 4 groups of mice that received 1×10¹¹ particles ofAAV virus containing various DNA constructs 30 minutes after injectionof luciferin, the substrate for luciferase. FIG. 1A shows luciferaseexpression in various tissues, predominantly in lungs, liver and muscle,in Group 1 mice before (“Pre”) and after (“Post”) rapamycinadministration. FIG. 11B shows luciferase expression, predominantly inliver and muscle in Group 2 mice before (“Pre”) and after (“Post”)rapamycin administration. FIG. 11C shows luciferase expressionpredominantly in liver and muscle after (“Post”) rapamycinadministration, and shows that there is no luciferase expression before(“Pre”) rapamycin administration in Group 3 mice. FIG. 11D showsluciferase expression is restricted to the liver (“Post”) rapamycinadministration and shows that there is no luciferase expression before(“Pre”) rapamycin administration.

FIGS. 12A-C. PITA DNA constructs for treating AMD. FIG. 12A shows a DNAconstruct comprising a transgene unit that encodes a soluble VEGFreceptor, sFlt-1. FIG. 12B shows a bicistronic DNA construct comprisingAvastin IgG heavy chain (AvastinH) and light chain (AvastinL) regulatedby IRES. FIG. 12C shows a bicistronic DNA construct comprising AvastinIgG heavy chain (AvastinH) and light chain (AvastinL) separated by a T2Asequence.

FIGS. 13A-B. PITA DNA constructs for treating Liver Metabolic Disease.FIG. 13A shows a PITA DNA construct for treating hemophilia A and/or B,containing a transgene unit comprising Factor IX. FIG. 13B shows a DNAconstruct for delivery of shRNA targeting the IRES of HCV.

FIGS. 14A-B. PITA DNA constructs for treating Heart Disease. FIG. 14Ashows a PITA DNA construct for treating congestive heart failure,containing a transgene unit comprising insulin like growth factor(IGF1). FIG. 14B shows a PITA DNA construct for treating congestiveheart failure, containing a transgene unit comprising hepatocyte growthfactor (HGF).

FIG. 15. PITA DNA construct for a CNS disease. FIG. 15 shows a PITA DNAconstruct for treating Alzheimer's disease, containing a transgene unitcomprising nerve growth factor (NGF).

FIG. 16. PITA System for HIV treatment. FIG. 16 shows a PITA DNAconstruct containing a transgene unit comprising the heavy and lightchains of an HIV antibody and a PITA DNA construct containing anablation unit and a dimerizable TF domain unit. FIG. 16 also shows thata rapamycin analog (rapalog) can induce expression of the ablator, cre,to ablate the transgene (heavy and light chains of an HIV antibody) fromthe PITA DNA construct containing a transgene unit.

FIG. 17. Illustration of one embodiment of the PITA system. FIG. 17shows a transgene unit encoding a therapeutic antibody that is inoperative association with a constitutive promoter, an ablation unitencoding an endonuclease that is in operative association with atranscription factor inducible promoter, and a dimerizable TF domainunit, with each transcription factor domain fusion sequence in operativeassociation with a constitutive promoter. Prior to administration ofrapamycin or a rapalog, there is baseline expression of the therapeuticantibody and of the two transcription factor domain fusion proteins.Upon rapamycin administration, the dimerized transcription factorinduces expression of the endonuclease, which cleaves the endonucleaserecognition domain in the transgene unit, thereby ablating transgeneexpression.

FIGS. 18A-18B are bar charts illustrating that wild-type FokI effectiveablated expression of a transgene when a DNA plasmid containing atransgene containing ablation sites for FokI was cotransfected intotarget cells with a plasmid encoding the FokI enzyme. FIG. 18A, bar 1represents 50 ng pCMV.Luciferase, bar 2 represents 50 ng pCMV.Luciferase+200 ng pCMV.FokI, bar 3 represents 50 ng pCMV.Luciferase +transfectedFokI protein, bar 4 represents transfected FokI protein alone; bar 5represents untransfected controls. FIG. 18B, bar 1 represents 50 ngpCMV.Luc alone, subsequent bars represent increasing concentrations of aZFHD-FokI expression plasmid (6.25, 12.5, 25, 50, and 100 ng)cotransfected with pCMV.Luciferase. This study is described in Example11A.

FIGS. 19A-B are bar charts illustrating that a chimeric engineeredenzyme tethered to a non-cognate recognition site on the DNA by the zincfinger homeodomain effectively ablates expression of a transgene. FIG.19A compares increasing concentrations of an expression plasmid encodingun-tethered FokI (6.25 ng, 12.5 ng, 25 ng, 50 ng and 100 ng)co-transfected with pCMV.luciferase. The first bar provides a positivecontrol of 50 ng pCMV.Luc alone. FIG. 19B compares increasingconcentrations of an expression plasmid encoding FokI tethered to DNAvia fusion with the zinc finger homeodomain (6.25 ng, 12.5 ng, 25 ng, 50ng and 100 ng) co-transfected with pCMV.luciferase. The first barprovides a control of 50 ng pCMV.Luc alone. This study is described inExample 11B.

FIGS. 20A-B are bar charts illustrating that the DNA binding specificityof chimeric FokI can be reproducible changed by fusion with variousclasses of heterologous DNA binding domains and ablation of targettransgene can be further improved by the additional of a heterologousnuclear localization signal (NLS). FIG. 20A illustrates the results ofco-transfection of pCMV.Luciferase with increasing concentrations of anexpression plasmid encoding FokI tethered to DNA via an HTH fusion(6.25, 12.5, 25, 50, and 100 ng). The first bar is a control showing 50ng pCMV.Luciferase alone. FIG. 20B illustrates the results ofco-transfection of pCMV.Luciferase with increasing concentrations of anexpression plasmid encoding an HTH-FokI fusion, which further has a NLSat its N-terminus (6.25, 12.5, 25, 50, and 100 ng). The first bar is acontrol showing 50 ng pCMV.Luciferase alone. This study is described inExample 11C.

FIG. 21 is a flow chart of a method for selecting a unique nucleic acidsequence for use in an ablation recognition site as described herein.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to gene therapy systems designed for thedelivery of a therapeutic product to a subject usingreplication-defective virus composition(s) engineered with a built-insafety mechanism for ablating the therapeutic gene product, eitherpermanently or temporarily, in response to a pharmacologicalagent—preferably an oral formulation, e.g., a pill.

In the PITA system, one or more replication-defective viruses are usedin a replication-defective virus composition in which the viralgenome(s) have been engineered to contain: (a) a first transcriptionunit that encodes a therapeutic product in operative association with apromoter that controls transcription, said unit containing at least oneablation recognition site; and (b) a second transcription unit thatencodes an ablator (or a fragment thereof as part of a fusion proteinunit) specific for the ablation recognition site in operativeassociation with a promoter that induces transcription in response to apharmacological agent. Any pharmacological agent that specificallydimerizes the domains of the selected binding domain can be used. In oneembodiment, rapamycin and its analogs referred to as “rapalogs” can beused.

A viral genome containing a first transcription unit may contain two ormore of the same ablation recognition site or two or more differentablation recognition sites (i.e., which are specific sites for adifferent ablator than that which recognizes the other ablationrecognition site(s)). Whether the same or different, such two or moreablation recognition sites may be located in tandem to one another, ormay be located in a position non-contiguous to the other. Further, theablation recognition site(s) may be located at any position relative thecoding sequence for the transgene, i.e., within the transgene codingsequence, 5′ to the coding sequence (either immediately 5′ or separatedby one or more bases, e.g., upstream or downstream of the promoter) or3′ to the coding sequence (e.g., either immediately 3′ or separated byone or more bases, e.g., upstream of the poly A sequence).

An ablator is any gene product, e.g., translational or transcriptionalproduct, that specifically recognizes/binds to either (a) the ablationrecognition site(s) (ARS) of the transgene unit and cleaves or excisesthe transgene; or (b) the ablation recognition RNA sequence (ARRS) ofthe transcribed transgene unit and cleaves or inhibits translation ofthe mRNA transcript. As described herein, an ablator may be selectedfrom the group consisting of: an endonuclease, a recombinase, ameganuclease, a zinc finger endonuclease, or a novel chimericendonuclease as described herein (comprising a catalytic domain and amulti-zinc finfer domain). These ablators are designed to binds to theablation recognition site in the first transcription unit and excises orablates DNA and an interfering RNA, a ribozyme, or an antisense thatablates the RNA transcript of the first transcription unit, orsuppresses translation of the RNA transcript of the first transcriptionunit. In one specific embodiment, the ablator is Cre (which has as itsablation recognition site loxP), or the ablator is FLP (which has as itsablation recognition site FRT). In one embodiment, an endonuclease isselected which functions independently of ATP hydrolysis. Examples ofsuch ablators may include a Type II S endonuclease (e.g., FokI), NaeI,and intron endonucleases (such as e.g., I-TevI), integrases (catalyzeintegration), serine recombinases (catalyze recombination), tyrosinerecombinases, invertases (e.g. Gin) (catalyze inversion), resolvases,(e.g., Tn3), and nucleases that catalyze translocation, resolution ,insertion, deletion, degradation or exchange.

For permanent shut down of the therapeutic transgene, the ablator can bean endonuclease that binds to the ablation recognition site(s) in thefirst transcription unit and ablates or excises the transgene. Wheretemporary shutdown of the transgene is desired, an ablator should bechosen that binds to the ablation recognition site(s) in the RNAtranscript of the therapeutic transgene and ablates the transcript, orinhibits its translation. In this case, interfering RNAs, ribozymes, orantisense systems can be used. The system is particularly desirable ifthe therapeutic transgene is administered to treat cancer, a variety ofgenetic disease which will be readily apparent to one of skill in theart, or to mediate host immune response.

Expression of the ablator may be controlled by one or more elements,including, e.g., an inducible promoter and/or by use of a chimericablator that utilizes a homodimer or heterodimer fusion protein system,such as are described herein. Where use of a homodimer system isselected, expression of the ablator is controlled by an induciblepromoter. Where use of heterodimer system is selected, expression of theablator is controlled by additional of a pharmacologic agent andoptionally, a further inducible promoter for one or both of the fusionproteins which form the heterodimer system. In one embodiment, a homo-and hetero-dimizerable ablator is selected to provide an additionallayer for safety to constructs with transcription factor regulators.These systems are described in more detail later in this specification.

Any virus suitable for gene therapy may be used, including but notlimited to adeno-associated virus (“AAV”); adenovirus; herpes virus;lentivirus; retrovirus; etc. In preferred embodiments, thereplication-defective virus used is an adeno-associated virus (“AAV”).AAV1, AAV6, AAV7, AAV8, AAV9 or rh10 being particularly attractive foruse in human subjects. Due to size constraints of the AAV genome forpackaging, the transcription units can be engineered and packaged in twoor more AAV stocks. Whether packaged in one viral stock which is used asa virus composition according to the invention, or in two or more viralstocks which form a virus composition of the invention, the viral genomeused for treatment must collectively contain the first and secondtranscription units encoding the therapeutic transgene and the ablator;and may further comprise additional transcription units. For example,the first transcription unit can be packaged in one viral stock, andsecond, third and fourth transcription units packaged in a second viralstock. Alternatively, the second transcription unit can be packaged inone viral stock, and the first, third and fourth transcription unitspackaged in a second viral stock. While useful for AAV due to sizecontains in packaging the AAV genome, other viruses may be used toprepare a virus composition according to the invention. In anotherembodiment, the viral compositions of the invention, where they containmultiple viruses, may contain different replication-defective viruses(e.g., AAV and adenovirus).

In one embodiment, a virus composition according to the inventioncontains two or more different AAV (or another viral) stock, in suchcombinations as are described above. For example, a virus compositionmay contain a first viral stock comprising the therapeutic gene withablator recognition sites and a first ablator and a second viral stockcontaining an additional ablator(s). Another viral composition maycontain a first virus stock comprising a therapeutic gene and a fragmentof an ablator and a second virus stock comprising another fragment of anablator. Various other combinations of two or more viral stocks in avirus composition of the invention will be apparent from the descriptionof the components of the present system.

In one embodiment, a composition contains one or more AAV vectors in asystem for delivery of a therapeutic product having a controlledtransgene expression ablation system. At least one AAV vector in thecompostion contains a nucleic acid molecule comprising: (i) a nucleicacid sequence encoding a therapeutic product operably linked to apromoter that controls transcription; and (ii) at least one endonucleaseablation site which comprises a sequence of at least 30 nucleic acidbase pairs which are specifically and selectively recognized by at leastten (10×) zinc fingers, said at least one endonuclease ablation sitebeing located at least 5′ to the sequence encoding the therapeuticproduct; and (b) at least one ablator which comprises a chimericendonuclease comprising at least ten copies of a zinc finger domainlinked to a functional endonuclease catalytic domain in operativeassociation with a promoter, wherein transcription and/or ablationactivity is induced in response to a pharmacological agent, said atleast ten (10×) zinc finger domain specifically and selectivelyrecognizing said at least about 30 base pair sequence in said at leastone endonuclease ablation site and comprising at least 10 independentlyselected recognition helices. In one embodiment, the endonucleasecatalytic domain is a FokI catalytic domain. In a further embodiment,the ablator (b) is controlled by a cassette that is activated by atranscription factor following being dimerized by a pharmacologic agent,said cassette comprising two transcription units, wherein one of saidtwo transcription units encoding the DNA binding domain of thetranscription factor fused to a binding domain for the pharmacologicalagent in operative association with a first promoter; and a second ofsaid two transcription units encoding the activation domain of thetranscription factor fused to a binding domain for the pharmacologicalagent in operative association with a second promoter. Anypharmacological agent that specifically dimerizes the domains of theselected binding domain can be used. In one embodiment, rapamycin andits analogs referred to as “rapalogs” can be used.

In order to conserve space within the viral genome(s), bicistronictranscription units can be engineered. For example, transcription unitsthat can be regulated by the same promoter, e.g., the third and fourthtranscription units (and where applicable, the first transcription unitencoding the therapeutic transgene) can be engineered as a bicistronicunit containing an IRES (internal ribosome entry site) or a 2A peptide,which self-cleaves in a post-translational event (e.g., furin-2A), andwhich allows coexpression of heterologous gene products by a messagefrom a single promoter when the transgene (or an ablator codingsequence) is large, consists of multi-subunits, or two transgenes areco-delivered, recombinant AAV (rAAV) carrying the desired transgene(s)or subunits are co-administered to allow them to concatamerize in vivoto form a single vector genome. In such an embodiment, a first AAV maycarry an expression cassette which expresses a single transgene and asecond AAV may carry an expression cassette which expresses a differenttransgene for co-expression in the host cell. However, the selectedtransgene may encode any biologically active product or other product,e.g., a product desirable for study. A single promoter may directexpression of an RNA that contains, in a single open reading frame(ORF), two or three heterologous genes (e.g., the third and fourthtranscription units, and where applicable, the first transcription unitencoding the therapeutic transgene) separated from one another bysequences encoding a self-cleavage peptide (e.g., 2A peptide, T2A) or aprotease recognition site (e.g., furin). The ORF thus encodes a singlepolyprotein, which, either during (in the case of T2A) or aftertranslation, is cleaved into the individual proteins. These IRES andpolyprotein systems can be used to save AAV packaging space, they canonly be used for expression of components that can be driven by the samepromoter.

The invention also relates to DNA constructs used to engineer cell linesfor the production of the replication-defective virus compositions;methods for producing and manufacturing the replication-defective viruscompositions; expression in a variety of cell types and systems,including plants, bacteria, mammalian cells, etc., and methods oftreatment using the replication-defective virus compositions for genetransfer, including veterinary treatment (e.g., in livestock and othermammals), and for in vivo or ex vivo therapy, including gene therapy inhuman subjects.

5.1. Transgene Ablation System

The present invention provides a Pharmacologically Induced TransgeneAblation (PITA) System designed for the delivery of a transgene(encoding a therapeutic product—protein or RNA) usingreplication-defective virus compositions engineered with a built-insafety mechanism for ablating the therapeutic gene product, eitherpermanently or temporarily, in response to a pharmacologicalagent—preferably an oral formulation, e.g., a pill containing a smallmolecule that induces expression of the ablator specific for thetransgene or its transcription product. However, other routes ofdelivery for the pharmacologic agent may be selected.

In the PITA system, one or more replication-defective viruses are usedin which the viral genome(s) have been engineered to contain a transgeneunit (described in Section 5.1.1 herein) and an ablation unit (describedin Section 5.1.2 herein). In particular, one or morereplication-defective viruses are used in which the viral genome(s) havebeen engineered to contain (a) a first transcription unit that encodes atherapeutic product in operative association with a promoter thatcontrols transcription, said unit containing at least one ablationrecognition site (a transgene unit); and (b) a second transcription unitthat encodes an ablator specific for the ablation recognition site inoperative association with a promoter that induces transcription inresponse to a pharmacological agent (an ablation unit).

In one embodiment, the PITA system is designed such that the viralgenome(s) of the replication-defective viruses are further engineered tocontain a dimerizable domain unit (described in Section 5.1.3). In oneembodiment, by delivering a dimerizable TF domain unit, target cells aremodified to co-express two fusion proteins: one containing a DNA-bindingdomain (DBD) of the transcription factor that binds the induciblepromoter controlling the ablator and the other containing atranscriptional activation domain (AD) of the transcription factor thatactivates the inducible promoter controlling the ablator, each fused todimerizer binding domains (described in Section 5.1.3). Addition of apharmacological agent, or “dimerizer” (described in Section 5.1.4) thatcan simultaneously interact with the dimerizer binding domains presentin both fusion proteins results in recruitment of the AD fusion proteinto the regulated promoter, initiating transcription of the ablator. See,e.g., the Ariad ARGENT® system described in U.S. Pat. No. 5,834,266 andU.S. Pat. No. 7,109,317, each of which is incorporated by referenceherein in its entirety. By using dimerizer binding domains that have noaffinity for one another in the absence of ligand and an appropriateminimal promoter, transcription is made absolutely dependent on theaddition of the dimerizer.

To this end, the viral genome(s) of the replication-defective virusescan be further engineered to contain a third and a fourth transcriptionunit (a dimerizable TF domain unit), each encoding a dimerizable domainof a transcription factor that regulates the inducible promoter of theablator in second transcription unit, in which: (c) the thirdtranscription unit encodes the DNA binding domain of the transcriptionfactor fused to a binding domain for the pharmacological agent inoperative association with a constitutive promoter; and (d) the fourthtranscription unit encodes the activation domain of the transcriptionfactor fused to a binding domain for the pharmacological agent inoperative association with a promoter. In one embodiment, each componentof the dimerizable TF domain is expressed under constitutive promoter.In another embodiment, at least one component of the dimerizable TFdomain unit is expressed under an inducible promoter.

One embodiment of the PITA system is illustrated in FIG. 21, which showsa transgene unit encoding a therapeutic antibody that is in operativeassociation with a constitutive promoter, an ablation unit encoding anendonuclease that is in operative association with a transcriptionfactor inducible promoter, and a dimerizable TF domain unit, with eachtranscription factor domain fusion sequence in operative associationwith a constitutive promoter. Prior to administration of rapamycin or arapalog, there is baseline expression of the therapeutic antibody and ofthe two transcription factor domain fusion proteins. Upon rapamycinadministration, the dimerized transcription factor induces expression ofthe endonuclease, which cleaves the endonuclease recognition domain inthe transgene unit, thereby ablating transgene expression.

In one embodiment, the replication-defective virus used in the PITAsystem is an adeno-associated virus (“AAV”) (described in Section5.1.5). AAV1, AAV6, AAV7, AAV8, AAV9 or rh10 are particularly attractivefor use in human subjects. Due to size constraints of the AAV genome forpackaging, the transcription units can be engineered and packaged in twoor more AAV stocks. For example, the first transcription unit can bepackaged in one AAV stock, and the second, third and fourthtranscription units packaged in a second AAV stock. Alternatively, thesecond transcription unit can be packaged in one AAV stock, and thefirst, third and fourth transcription units packaged in a second AAVstock.

5.1.1. Transgene Unit

In the PITA system, one or more replication-defective viruses are usedin which the viral genome(s) have been engineered to contain a transgeneunit. As used herein, the term “transgene unit” refers to a DNA thatcomprises: (1) a DNA sequence that encodes a transgene; (2) at least oneablation recognition site (ARS) contained in a location which disruptstransgene expression, including, within or flanking the transgene or itsexpression control elements (e.g., upstream or downstream of thepromoter and/or upstream of the polyA signal); and (3) a promotersequence that regulates expression of the transgene. The DNA encodingthe transgene can be genomic DNA, cDNA, or a cDNA that includes one ormore introns which e.g., may enhance expression of the transgene. Insystems designed for removal of the transgene, the ARS used is onerecognized by the ablator (described in Section 5.1.2) that ablates orexcises the transgene, e.g., an endonuclease recognition sequenceincluding but not limited to a recombinase (e.g., the Cre/loxP system,the FLP/FRT system), a meganuclease (e.g., I-eSce1 system), anartificial restriction enzyme system or another artificial restrictionenzyme system, such as the zinc finger nuclease, or a restriction enzymespecific for a restriction site that occurs rarely in the human genome,and the like. To repress expression of the transgene, the ARS can encodean ablation recognition RNA sequence (ARRS), i.e., an RNA sequencerecognized by the ablator that ablates the transcription product of thetransgene or translation of its mRNA, e.g., a ribozyme recognitionsequence, an RNAi recognition sequence, or an antisense recognitionsequence.

Examples of transgenes that can be engineered in the transgene units ofthe present invention includes, but are not limited to a transgene thatencodes: an antibody or antibody fragment that neutralizes HIVinfectivity, a therapeutic antibody such as VEGF antibody, TNF-αantibody (e.g., infliximab, adalimumab), an EGF-R antibody, basiliximab,cetuximab, infliximab, rituxumab, alemtuzumab-CLL, daclizumab,efalizumab, omalizumab, pavilizumab, trastuzumab, gemtuzumab,adalimumab, or an antibody fragment of any of the foregoing therapeuticantibodies; soluble vascular endothelial growth factor receptor-1(sFIt-1), soluble TNF-a receptor (e.g., etanercept), Factor VIII, FactorIX, insulin, insulin like growth factor (1GF), hepatocyte growth factor(RGF), heme oxygenase-1 (RO-1), nerve growth factor (NGF), beta-IFN,IL-6, anti-EGFR antibody, interferon (IFN), IFN beta-1a, anti-CD20antibody, glucagon-like peptide-1 (GLP-1), anti-cellular adhesionmolecule, a4-integrin antibody, glial cell line-derived neurotrophicfactor (GDNF), aromatic L-amino acid decarboxylase (ADCC), brain-derivedneurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), galanin,neuropeptide Y (NPY), a TNF antagonist, chemokines from the IL-8 family,BC12, IL-10, a therapeutic siRNA, a therapeutic u6 protein, endostatin,plasminogen or a fragment thereof, TIMP3, VEGF-A, RIFI alpha, PEDF, orIL-1 receptor antagonist.

The transgene can be under the control of a constitutive promoter, aninducible promoter, a tissue-specific promoter, or a promoter regulatedby physiological cues. Examples of constitutive promoters suitable forcontrolling expression of the therapeutic products include, but are notlimited to human cytomegalovirus (CMV) promoter, the early and latepromoters of simian virus 40 (SV40), U6 promoter, metallothioneinpromoters, EF1a promoter, ubiquitin promoter, hypoxanthinephosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase(DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerolkinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutasepromoter, the β-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA86: 10006-10010 (1989>>, the long terminal repeats (LTR) of MoloneyLeukemia Virus and other retroviruses, the thymidine kinase promoter ofHerpes Simplex Virus and other constitutive promoters known to those ofskill in the art.

Inducible promoters suitable for controlling expression of thetherapeutic product include promoters responsive to exogenous agents(e.g., pharmacological agents) or to physiological cues. These responseelements include, but are not limited to a hypoxia response element(HRE) that binds HIF-Iα and β, tetracycline response element (such asdescribed by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA89:5547-551); an ecdysone-inducible response element (No D et al., 1996,Proc. Natl. Acad. Sci. USA. 93 :3346-3351) a metal-ion response elementsuch as described by Mayo et al. (1982, Cell 29:99-108); Brinster et al.(1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol.5:1480-1489); a heat shock response element such as described by Noueret al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla.,pp 167-220, 1991); or a hormone response element such as described byLee et al. (1981, Nature 294:228-232); Hynes et al. (Proc. Natl. Acad.Sci. USA 78:2038-2042, 1981); Klock et al. (Nature 329:734-736, 1987);and Israel and Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and otherinducible promoters known in the art. Preferably the response element isan ecdysone-inducible response element, more preferably the responseelement is a tetracycline response element.

Examples of tissue-specific promoters suitable for use in the presentinvention include, but are not limited to those listed in Table 1 andother tissue-specific promoters known in the art.

TABLE 1 Tissue-specific promoters Tissue Promoter Liver TBG, A1AT HeartTroponin T (TnT) Lung CC10, SPC, FoxJ1 Central Nervous Synapsin,Tyrosine Hydroxylase, System/Brain CaMKII (Ca2+/calmodulin- dependentprotein kinase) Pancreas Insulin, Elastase-I Adipocyte Ap2, AdiponectinMuscle Desmin, MHC Endothelial cells Endothelin-I (ET-I), Flt-I RetinaVMD

For example, and not by way of limitation, the replication-defectivevirus compositions of the invention can be used to deliver a VEGFantagonist for treating accelerated macular degeneration in a humansubject; Factor VIII for treating hemophilia A in a human subject;Factor IX for treating hemophilia B in a human subject; insulin likegrowth factor (IGF) or hepatocyte growth factor (HGF) for treatingcongestive heart failure in a human subject; nerve growth factor (NGF)for treating a central nervous system disorder in a human subject; or aneutralizing antibody against HIV for treating HIV infection in a humansubject.

Still other useful therapeutic products include hormones and growth anddifferentiation factors including, without limitation, insulin,glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormonereleasing factor (GRF), follicle stimulating hormone (FSH), luteinizinghormone (LH), human chorionic gonadotropin (hCG), vascular endothelialgrowth factor (VEGF), angiopoietins, angiostatin, granulocyte colonystimulating factor (GCSF), erythropoietin (EPO), connective tissuegrowth factor (CTGF), basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin growth factors I and II(IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors α and β,interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses gene products suchas members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP 1,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin gene product [e.g., a mini- or micro-dystrophin]. Still otheruseful gene products include enzymes such as may be useful in enzymereplacement therapy, which is useful in a variety of conditionsresulting from deficient activity of enzyme. For example, enzymes thatcontain mannose-6-phosphate may be utilized in therapies for lysosomalstorage diseases (e.g., a suitable gene includes that encodingβ-glucuronidase (GUSH)).

5.1.2. Ablation Unit

The viral genome(s) of one or more replication-defective viruses used inthe PITA system are engineered to further contain an ablation unit orcoding sequences for an ablator, as defined here.

For permanent shut down of transgene expression, the ablator can be anendonuclease, including but not limited to a recombinase, ameganuclease, a zinc finger endonuclease or any restriction enzyme witha restriction site that rarely occurs in the human genome, that binds tothe ARS of the transgene unit and ablates or excises the transgene.Examples of such ablators include, but are not limited to the Cre/loxPsystem (Groth et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5995-6000);the FLP/FRT system (Sorrell et al., 2005, Biotechnol. Adv. 23, 431-469);meganucleases such as I-SceI which recognizes a specific asymmetric 18bp element (T AGGGAT AACAGGGT AAT (SEQ ID NO: 25)), a rare sequence inthe mammalian genome, and creates double strand breaks (Jasin, M., 1996,Trends Genet., 12,224-228); and artificial restriction enzymes (e.g., azinc finger nucleases generated by fusing a zinc finger DNA-bindingdomain to a DNA-cleavage domain that can be engineered to target ARSsequences unique to the mammalian genome (Miller et al., 2008, Proc.Natl. Acad. Sci. USA, 105: 5809-5814)). In one embodiment, the ablatoris a chimeric enzyme, which may be based on a homodimer or a heterodimerfusion protein.

Where temporary shutdown of the transgene is desired, an ablator shouldbe chosen that binds to the ARRS of the RNA transcript of the transgeneunit and ablates the transcript, or inhibits its translation. Examplesof such ablators include, but are not limited to interfering RNAs(RNAi), ribozymes such as riboswitch (Bayer et al., 2005, NatBiotechnol. 23(3):337-43), or antisense oligonucleotides that recognizean ARRS. RNAi, ribozymes, and antisense oligonucleotides that recognizean ARRS can be designed and constructed using any method known to thoseof skill in the art. This system is particularly desirable if thetherapeutic transgene is administered to treat cancer or to mediate hostimmune response.

In one embodiment, expression of the ablator must be controlled by aninducible promoter that provides tight control over the transcription ofthe ablator gene e.g., a pharmacological agent, or transcription factorsactivated by a pharmacological agent or in alternative embodiments,physiological cues. Promoter systems that are non-leaky and that can betightly controlled are preferred. Inducible promoters suitable forcontrolling expression of the ablator are e.g., response elementsincluding but not limited to a tetracycline (tet) response element (suchas described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA89:5547-551); an ecdysone-inducible response element (No D et al., 1996,Proc. Natl. Acad. Sci. USA. 93:3346-3351) a metal-ion response elementsuch as described by Mayo et al. (1982, Cell. 29:99-108); Brinster etal. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol.5: 1480-1489); a heat shock response element such as described by Noueret al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla.,pp 167-220, 1991); or a hormone response element such as described byLee et al. (1981, Nature 294:228-232); Hynes et al. (1981, Proc. Natl.Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736);and Israel & Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and otherinducible promoters known in the art. Using such promoters, expressionof the ablator can be controlled, for example, by the Tet-on/off system(Gossen et ai., 1995, Science 268:1766-9; Gossen et ai., 1992, Proc.Natl. Acad. Sci. USA., 89(12):5547-51); the TetR-KRAB system (UrrutiaR., 2003, Genome Biol., 4(10):231; Deuschle U et aI., 1995, Mol CellBiol. (4):1907-14); the mifepristone (RU486) regulatable system(Geneswitch; Wang Y et ai., 1994, Proc. Natl. Acad. Sci. USA.,91(17):8180-4; Schillinger et aI, 2005, Proc. Natl. Acad. Sci.USA.102(39):13789-94); the humanized tamoxifen-dep regulatable system(Roscilli et aI, 2002, Mol. Ther. 6(5):653-63); and the ecdysone-depregulatable system (Rheoswitch; Karns et al., 2001, BMC Biotechnol. 1:11; Palli et al., 2003, Eur J Biochem. 270(6):1308-15) to name but afew.

A chimeric enzyme may be controlled by a constitutive or an induciblepromoter. In one embodiment, the system utilizes a chimericendonuclease, wherein the nuclease has at least two domains, i.e., acatalytic domain and a sequence specific DNA binding domain, each ofwhich are expressed under separately controlled promoters and which areoperatively linked. When the two domains are expressed at the same time,the products of the two domains form a chimeric endonuclease. Typically,separate transcription units containing each of domains linked to a DNAbinding domain are provided. Such DNA binding domains include, forexample, zinc finger motifs, homeo domain motifs, HMG-box domains, STATproteins, B3, helix-loop-helix, winged helix-turn-helix, leucine zipper,helix-turn-helix, winged helix, POU domains, DNA binding domains ofrepressors, DNA binding domains of oncogenes and naturally occurringsequence-specific DNA binding proteins that recognize >6 base pairs.[U.S. Pat. No. 5,436,150, issued Jul. 25, 1995].

In one embodiment, the expression of the ablator is under the control ofan inducible promoter that is regulated by the dimerizable transcriptionfactor domains described in Section 5.1.3. An example of such aninducible promoter includes, but is not limited to a GAL4 binding siteminimum promoter, which is responsive to a GAL4 transcription factor. AGAL4 DNA binding domain or transactivation domain can also be fused to asteroid receptor, such as the ecdysone receptor (EcR). Still othersuitable inducible promoters, such as are described herein, may beselected.

5.1.3. Dimerizable Transcription Factor Domain Unit

In one embodiment, the PITA system is designed such that the viralgenome(s) of the replication-defective viruses are further engineered tocontain a dimerizable units which are heterodimer fusion proteins. Theseunits may be a dimerizable TF unit as defined herein or anotherdimerizable fusion protein unit (e.g., part of a chimeric enzyme). Insuch an instance, a dimerizer is used (see Section 5.1.4), which bindsto the dimerizer binding domains and dimerizes (reversibly cross-links)the DNA binding domain fusion protein and the activation domain fusionprotein, forming a bifunctional transcription factor. See, e.g., theAriad ARGENT™ system, which is described in U.S. Publication No.2002/0173474, U.S. Publication No. 200910100535, U.S. Pat. No.5,834,266, U.S. Pat. No. 7,109,317, U.S. Pat. No. 7,485,441, U.S. Pat.No. 5,830,462, U.S. Pat. No. 5,869,337, U.S. Pat. No. 5,871,753, U.S.Pat. No. 6,011,018, U.S. Pat. No. 6,043,082, U.S. Pat. No. 6,046,047,U.S. Pat. No. 6,063,625, U.S. Pat. No. 6,140,120, U.S. Pat. No.6,165,787, U.S. Pat. No. 6,972,193, U.S. Pat. No. 6,326,166, U.S. Pat.No. 7,008,780, U.S. Pat. No. 6,133,456, U.S. Pat. No. 6,150,527, U.S.Pat. No. 6,506,379, U.S. Pat. No. 6,258,823, U.S. Pat. No. 6,693,189,U.S. Pat. No. 6,127,521, U.S. Pat. No. 6,150,137, U.S. Pat. No.6,464,974, U.S. Pat. No. 6,509,152, U.S. Pat. No. 6,015,709, U.S. Pat.No. 6,117,680, U.S. Pat. No. 6,479,653, U.S. Pat. No. 6,187,757, U.S.Pat. No. 6,649,595, U.S. Pat. No. 6,984,635, U.S. Pat. No. 7,067,526,U.S. Pat. No. 7,196,192, U.S. Pat. No. 6,476,200, U.S. Pat. No.6,492,106, WO 94/18347, WO 96/20951, WO 96/06097, WO 97/31898, WO96/41865, WO 98/02441, WO 95/33052, WO 99110508, WO 99110510, WO99/36553, WO 99/41258,WO 01114387, ARGENT™ Regulated TranscriptionRetrovirus Kit, Version 2.0 (9109102), and ARGENT™ RegulatedTranscription Plasmid Kit, Version 2.0 (9109/02), each of which isincorporated herein by reference in its entirety.

In one embodiment, by delivering a dimerizable unit, target cells aremodified to co-express two fusion proteins that are dimerized by thepharmacologic agent used: one containing a DNA-binding domain (DBD) ofthe transcription factor that binds the inducible promoter controllingthe ablator and the other containing a transcriptional activation domain(AD) of the transcription factor that activates the inducible promotercontrolling the ablator, each fused to dimerizer binding domains.Expression of the two fusion proteins may be constitutive, or as anadded safety feature, inducible. Where an inducible promoter is selectedfor expression of one of the fusion proteins, the promoter mayregulatable, but different from any other inducible or regulatablepromoters in the viral composition. Addition of a pharmacological agent,or “dimerizer” (described in Section 5.1.4) that can simultaneouslyinteract with the dimerizer binding domains present in both fusionproteins results in recruitment of the AD fusion protein to theregulated promoter, initiating transcription of the ablator. By usingdimerizer binding domains that have no affinity for one another in theabsence of ligand and an appropriate minimal promoter, transcription ismade absolutely dependent on the addition of the dimerizer. Suitably, areplication-defective virus composition of the invention may containmore than one dimerizable domain. The various replication-defectiveviruses in a composition may be of different stock, which providedifferent transcription units (e.g., a fusion protein to form adimerable unit in situ) and/or additional ablators.

Fusion proteins containing one or more transcription factor domains aredisclosed in WO 94/18317, PCT/US94/08008, Spencer et al, supra and Blauet al. (PNAS 1997 94:3076) which are incorporated by reference herein intheir entireties. The design and use of such fusion proteins forligand-mediated gene-knock out and for ligand-mediated blockade of geneexpression or inhibition of gene product function are disclosed inPCT/US95/10591. Novel DNA binding domains and DNA sequences to whichthey bind which are useful in embodiments involving regulatedtranscription of a target gene are disclosed, e.g., in Pomeranz et al,1995, Science 267:93 96. Those references provide substantialinformation, guidance and examples relating to the design, constructionand use of DNA constructs encoding analogous fusion proteins, targetgene constructs, and other aspects which may also be useful to thepractitioner of the subject invention.

Preferably the DNA binding domain, and a fusion protein containing it,binds to its recognized DNA sequence with sufficient selectivity so thatbinding to the selected DNA sequence can be detected (directly orindirectly as measured in vitro) despite the presence of other, oftennumerous other, DNA sequences. Preferably, binding of the fusion proteincomprising the DNA-binding domain to the selected DNA sequence is atleast two, more preferably three and even more preferably more than fourorders of magnitude greater than binding to anyone alternative DNAsequence, as measured by binding studies in vitro or by measuringrelative rates or levels of transcription of genes associated with theselected DNA sequence as compared to any alternative DNA sequences. Thedimerizable transcription factor (TF) domain units of the invention canencode DNA binding domains and activation domains of any transcriptionfactor known in the art. Examples of such transcription factors includebut are not limited to GAL4, ZFHD1, VPI6, and NF-KB (p65).

The dimerizer binding domain encoded by a dimerizable unit of theinvention can be any dimerizer binding domain described in U.S.Publication No. 2002/0173474, U.S. Publication No. 200910100535, U.S.Pat. No. 5,834,266, U.S. Pat. No. 7,109,317, U.S. Pat. No. 7,485,441,U.S. Pat. No. 5,830,462, U.S. Pat. No. 5,869,337, U.S. Pat. No.5,871,753, U.S. Pat. No. 6,011,018, U.S. Pat. No. 6,043,082, U.S. Pat.No. 6,046,047, U.S. Pat. No. 6,063,625, U.S. Pat. No. 6,140,120, U.S.Pat. No. 6,165,787, U.S. Pat. No. 6,972,193, U.S. Pat. No. 6,326,166,U.S. Pat. No. 7,008,780, U.S. Pat. No. 6,133,456, U.S. Pat. No.6,150,527, U.S. Pat. No. 6,506,379, U.S. Pat. No. 6,258,823, U.S. Pat.No. 6,693,189, U.S. Pat. No. 6,127,521, U.S. Pat. No. 6,150,137, U.S.Pat. No. 6,464,974, U.S. Pat. No. 6,509,152, U.S. Pat. No. 6,015,709,U.S. Pat. No. 6,117,680, U.S. Pat. No. 6,479,653, U.S. Pat. No.6,187,757, U.S. Pat. No. 6,649,595, U.S. Pat. No. 6,984,635, U.S. Pat.No. 7,067,526, U.S. Pat. No. 7,196,192, U.S. Pat. No. 6,476,200, U.S.Pat. No. 6,492,106, WO 94118347, WO 96/20951, WO 96/06097, WO 97/31898,WO 96/41865, WO 98/02441, WO 95/33052, WO 99/10508, WO 99110510, WO99/36553, WO 99/41258, WO 01114387, ARGENT™ Regulated TranscriptionRetrovirus Kit, Version 2.0 (Sep. 9, 2002), and ARGENT™ RegulatedTranscription Plasmid Kit, Version 2.0 (Sep. 9, 2002), each of which isincorporated herein by reference in its entirety.

A dimerizer binding domain that can be used in the PITA system is theimmunophilin FKBP (FK506-binding protein). FKBP is an abundant 12 kDacytoplasmic protein that acts as the intracellular receptor for theimmunosuppressive drugs FK506 and rapamycin. Regulated transcription canbe achieved by fusing multiple copies of FKBP to a DNA binding domain ofa transcription factor and an activation domain of a transcriptionfactor, followed by the addition of FK1012 (a homodimer of FK506; Ho, S.N., et al., 1996, Nature, 382(6594): 822-6); or simpler syntheticanalogs such as AP1510 (Amara, J. F., et aI., 1997, Proc. Natl. Acad.Sci. USA, 94(20): 10618-23). The potency of these systems can beimproved by using synthetic dimerizers, such as AP1889, with designed‘bumps’ that minimize interactions with endogenous FKBP (Pollock et al.,1999, Methods Enzymol, 1999.306: p. 263-81) Improved approaches based onheterodimerization, exploiting the discovery that FK506 and rapamycinnaturally function by bringing together FKBP with a second targetprotein. This allows the natural products themselves, or analogsthereof, to be used directly as dimerizers to control gene expression.

The structure of FKBP-FK506 complexed to calcineurin phosphatase(Griffith et aI, Cell, 82:507 522, 1995) has been reported. CalcineurinA (residues 12 394) was shown to be effective as a dimerizer bindingdomain using a three hybrid system in yeast using three FKBPs fused toGa14 and residues 12 to 394 of murine calcineurin A fused C-terminallyto the Ga14 activation domain (Ho, 1996 Nature. 382:822 826). Additionof FK506 activated transcription of a reporter gene in these cells. A“minimal” calcineurin domain termed a CAB, which is a smaller, moremanipulatable domain can be used as a dimerizer binding domain.

The DNA binding domain fusion protein and activation domain fusionprotein encoded by the dimerizable fusion protein units of the inventionmay contain one or more copies of one or more different dimerizerbinding domains The dimerizer binding domains may be N-terminal,C-terminal, or interspersed with respect to the DNA binding domain andactivation domain. Embodiments involving multiple copies of a dimerizerbinding domains usually have 2, 3 or 4 such copies. The various domainsof the fusion proteins are optionally separated by linking peptideregions which may be derived from one of the adjacent domains or may beheterologous.

As used herein, the term “variants” in the context of variants ofdimerizer binding domains refers to dimerizer binding domains thatcontain deletions, insertions, substitutions, or other modificationsrelative to native dimerizer binding domains, but that retain theirspecificity to bind to dimerizers. The variants of dimerizer bindingdomains preferably have deletions, insertions, substitutions, and/orother modifications of not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid residues. In a specific embodiment, the variant of adimerizer binding domain has the native sequence of a dimerizer bindingdomain as specified above, except that 1 to 5 amino acids are added ordeleted from the carboxy and or the amino end of the dimerizer bindingdomains (where the added amino acids are the flanking amino acid(s)present in the native dimerizer binding domains).

In order to conserve space within the viral genome(s), bicistronictranscription units can be engineered. For example, the third and fourthtranscription units can be engineered as a bicistronic unit containingan IRES (internal ribosome entry site), which allows coexpression ofheterologous gene products by a message from a single promoter.Altenatively, a single promoter may direct expression of an RNA thatcontains, in a single open reading frame (ORF), two or threeheterologous genes (e.g., the third and fourth transcription units)separated from one another by sequences encoding a self-cleavage peptide(e.g., T2A) or a protease recognition site (e.g., furin). The ORF thusencodes a single polyprotein, which, either during (in the case of T2A)or after translation, is cleaved into the individual proteins. It shouldbe noted, however, that although these IRES and polyprotein systems canbe used to save AAV packaging space, they can only be used forexpression of components that can be driven by the same promoter.

As illustrated in the examples below, various components of theinvention may include:

ITR: inverted terminal repeats (ITR) of AAV serotype 2 (168 bp). In oneembodiment, the AAV2 ITRs are selected to generate a pseudotyped AAV,i.e., an AAV having a capsid from a different AAV than that the AAV fromwhich the ITRs are derived.

CMV: full cytomegalovirus (CMV) promoter; including enhancer. CMV:minimal CMV promoter, not including enhancer. In one embodiment, thehuman CMV promoter and/or enhancer are selected.

FRB-TA fusion: fusion of dimerizer binding domain and an activationdomain of a transcription factor. The FRB fragment corresponds to aminoacids 2021-2113 of FRAP (FKBP rapamycin-associated protein, also knownas mTOR [mammalian target of rapamycin]), a phosphoinositide 3-kinasehomolog that controls cell growth and division. The FRAP sequenceincorporates the single point-mutation Thr2098Leu (FRAP_(L)) to allowuse of certain non-immunosuppressive rapamycin analogs (rapalogs). FRAPbinds to rapamycin (or its analogs) and FKBP and is fused to a portionof human NF-KB p65 (190 amino acids) as transcription activator.

ZFHD-FKBP fusion: fusion of a DNA binding domain and 1 copy of aDimerizer binding domain, 2 copies of drug binding domain (2xFKBP, or 3(3xFKBP) copies of drug binding domain Immunophilin FKBP (FK506-bindingprotein) is an abundant 12 kDa cytoplasmic protein that acts as theintracellular receptor for the immunosuppressive drugs FK506 andrapamycin. ZFHD is DNA binding domains composed of a zinc finger pairand a homeodomain. In another alternative, various other copy numbers ofa selected drug binding domain may be selected. Such fusion proteins maycontain N-terminal nuclear localization sequence from human c-Myc at the5′ and/or 3′ end.

Z8I: contains 8 copies of the binding site for ZFHD (Z8) followed byminimal promoter from the human interleukin-2 (1L-2) gene (SEQ ID NO:32). Variants of this may be used, e.g., which contain from 1 to about20 copies of the binding site for ZFHD followed by a promoter, e.g., theminimal promoter from IL-2 or another selected promoter.

Cre: Cre recombinase. Cre is a type I topoisomerase isolated frombacteriophage PI. Cre mediates site specific recombination in DNAbetween two loxP sites leading to deletion or gene conversion (1029 bp,SEQ ID NO: 33).

I-SceI: a member of intron endonuclease or homing endonuclease which isa large class of meganuclease (708 bp, SEQ ID NO: 34). They are encodedby mobile genetic elements such as introns found in bacteria and plants.I-SceI is a yeast endonuclease involved in an intron homing process.I-SceI recognizes a specific asymmetric 18 bp element, a rare sequencein mammalian genome, and creates double strand breaks. See, Jasin, M.(1996) Trends Genet., 12, 224-228.

hGH poly A: minimal poly adenylation signal from human GH (SEQ ID NO:35).

IRES: internal ribosome entry site sequence from ECMV(encephalomyocarditis virus) (SEQ ID NO: 36).

5.1.4. Dimerizers and Pharmacologic Agents

As used herein, the term “dimerizer” is a compound that can bind todimerizer binding domains of the TF domain fusion proteins (described inSection 5.1.3) and induce dimerization of the fusion proteins. Anypharmacological agent that dimerizes the domains of the transcriptionfactor, as assayed in vitro can be used. Preferably, rapamycin and itsanalogs referred to as “rapalogs” can be used. Any of the dimerizersdescribed in following can be used: U.S. Publication No. 2002/0173474,U.S. Publication No. 2009/0100535, U.S. Pat. No. 5,834,266, U.S. Pat.No. 7,109,317, U.S. Pat. No. 7,485,441, U.S. Pat. No. 5,830,462, U.S.Pat. No. 5,869,337, U.S. Pat. No. 5,871,753, U.S. Pat. No. 6,011,018,U.S. Pat. No. 6,043,082, U.S. Pat. No. 6,046,047, U.S. Pat. No.6,063,625, U.S. Pat. No. 6,140,120, U.S. Pat. No. 6,165,787, U.S. Pat.No. 6,972,193, U.S. Pat. No. 6,326,166, U.S. Pat. No. 7,008,780, U.S.Pat. No. 6,133,456, U.S. Pat. No. 6,150,527, U.S. Pat. No. 6,506,379,U.S. Pat. No. 6,258,823, U.S. Pat. No. 6,693,189, U.S. Pat. No.6,127,521, U.S. Pat. No. 6,150,137, U.S. Pat. No. 6,464,974, U.S. Pat.No. 6,509,152, U.S. Pat. No. 6,015,709, U.S. Pat. No. 6,117,680, U.S.Pat. No. 6,479,653, U.S. Pat. No. 6,187,757, U.S. Pat. No. 6,649,595,U.S. Pat. No. 6,984,635, U.S. Pat. No. 7,067,526, U.S. Pat. No.7,196,192, U.S. Pat. No. 6,476,200, U.S. Pat. No. 6,492,106, WO94118347, WO 96/20951, WO 96/06097, WO 97/31898, WO 96/41865, WO98/02441, WO 95/33052, WO 99/10508, WO 99/10510, WO 99/36553, WO99/41258, WO 01114387, ARGENT™ Regulated Transcription Retrovirus Kit,Version 2.0 (9109/02), and ARGENT™ Regulated Transcription Plasmid Kit,Version 2.0 (Sep. 9, 2002), each of which is incorporated herein byreference in its entirety.

Examples of dimerizers that can be used in the present inventioninclude, but are not limited to rapamycin, FK506, FKI012 (a homodimer ofFK506), rapamycin analogs (“rapalogs”) which are readily prepared bychemical modifications of the natural product to add a “bump” thatreduces or eliminates affinity for endogenous FKBP and/or FRAP. Examplesof rapalogs include, but are not limited to such as AP26113 (Ariad), AP1510 (Amara, J. F., et al.,1997, Proc Natl Acad Sci USA, 94(20):10618-23) AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856,AP1701, AP1861, AP1692 and AP1889, with designed ‘bumps’ that minimizeinteractions with endogenous FKBP.

Other dimerizers capable of binding to dimerizer binding domains or toother endogenous constituents may be readily identified using a varietyof approaches, including phage display and other biological approachesfor identifying peptidyl binding compounds; synthetic diversity orcombinatorial approaches (see e.g. Gordon et al, 1994, J Med Chem37(9):1233-1251 and 37(10):1385-1401); and DeWitt et al, 1993, PNAS USA90:6909-6913) and conventional screening or synthetic programs.Dimerizers capable of binding to dimerizer binding domains of interestmay be identified by various methods of affinity purification or bydirect or competitive binding assays, including assays involving thebinding of the protein to compounds immobilized on solid supports suchas pins, beads, chips, etc.). See e.g. Gordon et al., supra.

Generally speaking, the dimerizer is capable of binding to two (or more)protein molecules, in either order or simultaneously, preferably with aKd value below about 10⁻⁶ more preferably below about 10⁻⁷, even morepreferably below about 10⁻⁸, and in some embodiments below about 10⁻⁹ M.The dimerizer preferably is a non-protein and has a molecular weight ofless than about 5 kDa. The proteins so oligomerized may be the same ordifferent.

Various dimerizers are hydrophobic or can be made so by appropriatemodification with lipophilic groups. Particularly, dimerizers containinglinking moieties can be modified to enhance lipophilicity by includingone or more aliphatic side chains of from about 12 to 24 carbon atoms inthe linker moiety.

5.1.5. Generating Replication-Defective Virus Compositions

Any virus suitable for gene transfer (e.g., gene therapy) may be usedfor packaging the transcription units into one or more stocks ofreplication-defective virus, including but not limited toadeno-associated virus (“AAV”); adenovirus; alphavirus; herpesvirus;retrovirus (e.g., lentivirus); vaccinia virus; etc. Methods well knownin the art for packaging foreign genes into replication-defectiveviruses can be used to prepare the replication-defective virusescontaining the therapeutic transgene unit, the ablation unit, andoptionally (but preferably) the dimerizable transcription factor domainunit. See, for example, Gray & Samulski, 2008, “Optimizing gene deliveryvectors for the treatment of heart disease,” Expert Opin. Biol. Ther.8:911-922; Murphy & High, 2008, “Gene therapy for haemophilia,” Br. J.Haematology 140:479-487; Hu, 2008, “Baculoviral vectors for genedelivery: A review,” Current Gene Therapy 8:54-65; Gomez et al., 2008,“The poxvirus vectors MV A and NYV AC as gene delivery systems forvaccination against infectious diseases and cancer,” Current GeneTherapy 8:97-120.

In preferred embodiments, the replication-deficient virus compositionsfor therapeutic use are generated using an AAV. Methods for generatingand isolating AAVs suitable for gene therapy are known in the art. Seegenerally, e.g., Grieger & Samulski, 2005, “Adeno-associated virus as agene therapy vector: Vector development, production and clinicalapplications,” Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning etal., 2008, “Recent developments in adeno-associated virus vectortechnology,” J. Gene Med. 10:717-733; and the references cited below,each of which is incorporated herein by reference in its entirety.

Adeno-associated virus (genus Dependovirus, family Parvoviridae) is asmall (approximately 20-26 nm), non-enveloped single-stranded (ss) DNAvirus that infects humans and other primates. Adeno-associated virus isnot currently known to cause disease. Adeno-associated virus can infectboth dividing and non-dividing cells. In the absence of functionalhelper virus (for example, adenovirus or herpesvirus) AAV isreplication-defective. Adeno-associated viruses form episomalconcatamers in the host cell nucleus. In non-dividing cells, theseconcatamers remain intact for the life of the host cell. In dividingcells, AAV DNA is lost through cell division, since the episomal DNA isnot replicated along with the host cell DNA. However, AAV DNA may alsointegrate at low levels into the host genome.

The AAV genome is built of a ssDNA, either positive- or negative-sense,which is about 4.7 kilobases long. The genome of AAV as it occurs innature comprises inverted terminal repeats (ITRs) at both ends of theDNA strand, and two open reading frames (ORFs): rep and cap. The formeris composed of four overlapping genes encoding the Rep proteins that arerequired for the AAV life cycle, and the latter contains overlappingsequences that encode the capsid proteins (Cap): VP1, VP2, and VP3,which interact to form a capsid of an icosahedral symmetry.

The ITRs are 145 bases each, and form a hairpin that contributes toso-called “self-priming” that allows primase-independent synthesis ofthe second DNA strand. The ITRs also appear to be required for AAV DNAintegration into the host cell genome (e.g., into the 19th chromosome inhumans) and rescue from it, as well as for efficient encapsidation ofthe AAV DNA and assembly of AAV particles.

For packaging a transgene into virions, the ITRs are the only AAVcomponents required in cis in the same construct as the transgene. Thecap and rep genes can be supplied in trans. Accordingly, DNA constructscan be designed so that the AAV ITRs flank one or more of thetranscription units (i. e., the transgene unit, the ablator unit, andthe dimerizable transcription factor unit), thus defining the region tobe amplified and packaged—the only design constraint being the upperlimit of the size of the DNA to be packaged (approximately 4.5 kb).Adeno-associated virus engineering and design choices that can be usedto save space are described below.

Compositions and System Utilizing 10×Zinc Finger Constructs

The invention provides a method for vector-mediated delivery of atherapeutic product having a controlled gene expression ablation system.For convenience, the method is described in the context of an AAVvector. However, one of skill in the art can select another suitablevector (e.g., a replication-defective adenovirus, replication-defectivelentivirus, or other viral or genetic element) for use with thecompositions and methods described herein. Examples of viral and othervectors are described earlier and incorporated herein by reference. Inone embodiment, an AAV vector containing a nucleic acid moleculecomprising a nucleic acid sequence encoding a therapeutic or vaccinalproduct (or another gene or sequence of interest) operably linked to apromoter that controls transcription and at least one endonucleaseablation site which comprises a sequence of at least 30 nucleic acidbase pairs which are specifically and selectively recognized by aconstruct of at least ten (10×) zinc fingers. The at least oneendonuclease ablation site is located at least 5′ to the sequenceencoding the gene of interest (e.g., a transgene such as DNA, RNA,ribozyme, siRNA, shRNA, miRNA or protein, peptide, system's biologyengineered pathways, etc.). The method further involves use of at leastone ablator which comprises a chimeric endonuclease comprising at leastten zinc fingers linked to a functional endonuclease catalytic domain inoperative association with a promoter, wherein transcription and/orablation activity is induced in response to a pharmacological agent. Theat least ten (10×) zinc fingers specifically and selectively recognizesthe at least about 30 base pair sequence in the at least oneendonuclease ablation site and contains at least 10 independentlyselected recognition helices.

In one embodiment, the present invention utilizes chimeric endonucleasesas a safety measure necessary to destroy a gene of interest in genedelivery applications in vivo [including gene therapy, vaccines etc],for example when a transgene product is no longer required or exhibitstoxic/off target effects inside the organism. Thus, the chimericendonucleases of the invention have one or more of the followingfeatures. They intentionally target sequences that are not present inmammalian (e g., human) genomes (thus the term the “at least 30 bpunique sequence”), they have high in vivo selectivity to a targetlocation only, and they have minimal off-target and cytotoxic effects.Due to the fact that the endonucleases described previously in theliterature were used for a different purpose, i.e., for in situengineering of mammalian genomes to allow knock-out or correction(knock-in) of specific genes of interest, the inventors were required todevelop a new methodology to design a chimeric endonuclease with thefeatures required for the present inveniton, including designing longstretches, (i.e., in excess of 7, 8, 9, 10, 11, 12, or more zincfingers) and for selecting unique sequences that are not present withina target genome (e.g., a human or other mammalian subject).

In one embodiment, the zinc finger proteins are engineered tospecifically target a DNA sequence which is not part of the targetgenome in order to mitigate off-target effects and cytotoxicityassociated with customarily used designs.

For example, for delivery to humans, random sequences of at least 32base pair are generated in order to accommodate frame shifts, althoughsmaller sequences (e.g., at least 30 base pairs), could be selected.This size was selected because there are about 3.4×10⁹ base pairs inhuman genome. The number of all possible 15 base pair-long sequenceswith permutation of 4 nucleotides is 4¹⁵, which is same as approximately10⁹. Which means that for any given DNA sequence which is equal to orless than 15 base pairs, an identical subsequence within the humangenome can be found. Conversely, for any short DNA sequence, there willbe at least 15 positions that are identical to a piece of human DNAsequence. During initial assessment, no short sequences whose maximalsequence-identity with any human DNA sequence is less than 65% wereidentified.

Surprisingly, the first unique sequence randomly generated for use as anablation recognition site and tested was found to meet the desiredparameters: SEQ ID NO: 806-GGTCGATGTTCGCAACGTCGATCGTACGTGCA. Theexamples below illustrate the sequence being engineered into anexpression plasmid vector carrying the gene of interest and beingspecifically recognized by a chimeric endonuclease of the inventionwhich comprises at least ten zinc fingers. The sequences encoding theablation recognition site and the sequences of the chimeric endonucleasecan be engineered into suitable viral vectors (e.g., AAV vectors) usingconventional techniques in order to obtain the AAV vectors, compositionsand to perform the methods of the invention.

With reference to FIG. 21, a streamlined method for selecting a uniquesequence of the invention is provided. This involves randomly generate 2million short DNA sequences and selecting those with a sequence identityof less than (<) 20% to one another. The resulting sequences arecompared via BLAST using conventional parameters against the targetgenome (e.g., human) and those with an E-value of greater than 100 areselected. [An E-value is an Expect value. The statistical significancethreshold for reporting matches against database sequences; the defaultvalue is 10, meaning that 10 matches are expected to be found merely bychance, according to the stochastic model of Karlin and Altschul (1990).If the statistical significance ascribed to a match is greater than theExpect threshold, the match will not be reported. Lower Expectthresholds are more stringent, leading to fewer chance matches beingreported. Increasing the threshold shows less stringent matches.Fractional values are acceptable.] An exemplary sequence is compared forsimilarity against the human genome with a slide window. [For slidingwindows a window of 32 base pair slides over the nucleotides of thehuman genome. The window moves 1 nucleotide for each step. for everyposition of the window identity levels are calculated.] Any sequencewith a similarity greater than 70% is discarded. If the sequence has asimilarity of less than 70%, then it is analyzed against the targetgenome for the presence of any 8 consecutive base pairs and any having 8or more consecutive base pairs in common with the target sequence arediscarded. The remainder may be selected for use in an ablationrecognition site.

Thus, a unique sequence for an ablation recognition site of theinvention, has less than 70% identity with any subsequence in the humangenome, has no more than 8 adjacent identical positions with anysubsequence in the human genome. A zinc finger is thereafter designedwhich specifically targets this sequence as described herein. The uniquesequence can be read by different zinc finger designs which recognizedifferent reading frames such that it is possible for two distinct zincfingers to be be specific for a single unique at least 32 by sequence.The sequences may be 32, 33, 34, 35, 36, 38, 41, 44, or more sequencesin length,

In addition to the unique sequence identified above, other uniquesequences for use in the ablation recognition site were also generated:

SEQ ID NO: 801: SEQ ID NO: GGTCGGCGACGCGAATCGTCGATTGGCGTACSEQ ID NO: 802: SEQ ID NO: GGTCGGCGACGCGTATCGATTGGCGTAC andSEQ ID NO: 803: ACTATTCGCACGCCGTACGATAGTCGGCGCGA.

Once the specific ablation site sequence is selected, the ablator can beengineered to contain the zinc finger protein which will specificallyrecognize this unique at least 30 bp sequence within the ablation siteof the vector carrying the gene of interest.

In one embodiment, a chimeric endonuclease of the invention isengineered to contain at least the catalytic domain of an endonucleasefused to a zinc finger protein, optionally via a linker. The catalyticdomain may be located on the N-terminus of the zinc finger, theC-terminus of the zinc finger, or it may be located within the zincfinger (i.e., the catalytic domain is flanked on both its N- andC-terminus by zinc finger modules). Optionally, nuclear localizationsignals are included at the N- or C-terminus of the chimericendonuclease, or the N- or C-terminus of the zinc finger portion ofprotein. These elements are described elsewhere in the specification,which passages are incorporated herein by reference.

In one embodiment, the FokI catalytic domain is used without any otherfunctional sequence of the FokI endonuclease, i.e., the DNA bindingdomain is not present. Further, because of the manner in which thechimeric endonuclease described herein has been engineered, the knownFokI recognition site (5′-CATCC-3′) is not required. FokI catalyticdomain is a non-sequence specific endonuclease, once the ablatorrecognizes the ablation site, the location in the ablation recognitionsite where FokI cuts can be altered by the length of the optional linkersequence between the FokI catalytic domain and the zinc fingers.

For example, a linker sequence of five amino acids (e.g., SEQ ID NO:805, GTSGK) results in the FokI chimeric ablator cutting 6 bp directlyfollowing the zinc finger binding site. [The linker length is definedusing the convention set forth in Yuka Shimizu, et al, Bioorg Med ChemLett. 2009 Jul. 15; 19(14): 3970-3972]. Thus, the location of the cutmade by the FokI chimeric ablator can be adjusted by increasing ordecreasing the length of the linker. For example, other linkers for thechimeric ablator (amino acids of 0, 1, 2, 3, 4, 5 or 6 amino acids inlength) may be used in combination with spacers of 6, 7, 8, 9, or longeron the ablation site.

The zinc finger is a modular protein which binds DNA in reverseorientation (i.e., the N-terminus of the Zn finger binds starting at the3′ end of the sense strand). Typically, each module or finger uses aminoacids in positions: −1, 3, and 6 of its α-helix to target a specific3-bp recognition site on DNA (i.e., a “triplet”). These helices arespecific and selected for specific DNA triplets. Thus, for a 10× zincfinger protein, each of 10 zinc fingers (or modules) may beindependently selected to target the unique triplets present in the atleast 30 nucleic acid sequence of the invention.

The inventors have demonstrated specific and selective ablationaccording to the invention using a FokI catalytic domain fused to a 10×zinc finger protein specific for the unique sequence of the ablator.Given the description in the art of zinc fingers of significantlysmaller sizes (2-3) and a recent publication that zinc fingers largerthan 6× are not successful proteins [Yuka Shimizu, et al, Adding Fingersto an Engineered Zinc Finger Nuclease Can Reduce Activity, Biochemistry2011, 50, 5033-5041], this result was unexpected.

Once the size and sequence of the at least 30 base pair unique nucleicacid sequence of the ablation recognition site are known (e.g., at least30 base pairs where the vector is to be delivered to a human), one canengineer a zinc finger which provides for at least one module (1×) foreach triplet. Each zinc finger contains a recognition helix specific fora given triplet, in which sequence of recognition helix is engineered tofold into a conserved zinc finger structure in order to present theabout seven amino acid sequence in the proper helical form. Moreparticularly, applicants provide in the following Table 2 a selection ofillustrative conserved sequences which may be used to construct a zincfinger. In this table, the seven dashes represent the location in whichone of the seven amino acid helix recognition sequences may be insertedin order to construct a multi-(×)-zinc finger.

TABLE 2 Conserved Zinc Finger Sequences SEQ ID NO:CRCNECGKSFS-------HQRTH 706 QFACDICGRKFA-------HTKIH 707FACEVCGVRFT-------HMRKH 708 FACSWQDCNKKFA-------HYRTH 709FECKDCGKAFI-------HQRTH 710 FHCGYCEKSFS-------HIRTH 711FKCPVCGKAFR-------HQRTH 712 FLCQYCAQRFG-------HMKKSH 713FQCKTCQRKFS-------HTRTH 714 FQCNQCGASFT-------HIKLH 715FSCSWKGCERRFA-------HRRTH 716 GSQKPFQCRICMRNFS-------HIRTH 717HKCLECGKCFS-------HQRTH 718 MAERPFQCRICMRKFA-------HTKIH 719PGEKKFACPECPKRFM-------HIKTH 720 PGEKPFECKDCGKAFI-------HQRTH 721PGEKPFKCPVCGKAFR-------HQRTH 722 PGEKPFMCTWSYCGKRFT-------HKRTH 723PGEKPFQCKTCQRKFS-------HTRTH 724 PGEKPFQCNQCGASFT-------HIKLH 725PGEKPHICHIQGCGKVYG-------HLRWH 726 PGEKPYECDHCGKAFS-------HRRIH 727PGEKPYECDHCGKSFS-------HKRTH 728 PGEKPYECEKCGKAFN-------HKKSH 729PGEKPYECHDCGKSFR-------HRRIH 730 PGEKPYECKECGKAFS-------HQRIH 731PGEKPYECNYCGKTFS-------HQRIH 732 PGEKPYGCHLCGKAFS-------HEMIH 733PGEKPYICRKCGRGFS-------HQRTH 734 PGEKPYKCEECGKAFN-------HKIVH 735PGEKPYKCEECGKAFR-------HKIIH 736 PGEKPYKCEECGKAFT-------HKKIH 737PGEKPYKCGQCGKFYS-------HQKIH 738 PGEKPYKCHQCGKAFI-------HERTH 739PGEKPYKCKECGKAFN-------HHRIH 740 PGEKPYKCKECGQAFR-------HHKLH 741PGEKPYKCKQCGKAFG-------HGRTH 742 PGEKPYKCMECGKAFN-------HQRIH 743PGEKPYKCPDCGKSFS-------HQRTH 744 PGEKPYKCPECGKSFS-------HQRTH 745PGEKPYMCSECGRGFS-------HQRTH 746 PGEKPYRCEECGKAFR-------HKRIH 747PGEKPYRCKYCDRSFS-------HVRNIH 748 PGEKPYTCKQCGKAFS-------HETTH 749PGEKPYTCSDCGKAFR-------HRRTH 750 PGEKPYVCDVEGCTWKFA-------HKKRH 751PGEKPYVCRECGRGFR-------HKRTH 752 PGEKPYVCSKCGKAFT-------HQKIH 753PGERPFMCTWSYCGKRFT-------HKRTH 754 TGEKPFACDICGKKFA-------HTKIH 755TGEKPFACDICGRKFA-------HTKIH 756 TGEKPFQCRICMRNFS-------HIRTH 757TGSQKPFQCRICMRNFS-------HIRTH 758 VPERPFQCQICMRNFS-------HIRTH 759YACHLCAKAFI-------HEKTH 760 YACHLCGKAFT-------HEKTH 761YECDHCGKAFS-------HRRIH 762 YECDHCGKSFS-------HKRTH 763YECDVCGKTFT-------HQRTH 764 YECEKCGKAFN-------HKKSH 765YECHDCGKSFR-------HRRIH 766 YECKECGKAFS-------HQRIH 767YECNECGKAFA-------HQRIH 768 YECNECGKFFS-------HRRSH 769YECNTCRKTFS-------HQRTH 770 YECQDCGRAFN-------HKRTH 771YECVQCGKGFT-------HQRVH 772 YECVQCGKSYS-------HQRRH 773YGCHLCGKAFS-------HEMIH 774 YHCDWDGCGWKFA-------HYRKH 775YICRKCGRGFS-------HQRTH 776 YKCDECGKNFT-------HKRIH 777YKCEECGKAFN-------HKIVH 778 YKCEECGKAFR-------HKIIH 779YKCEECGKAFT-------HKKIH 780 YKCGQCGKFYS-------HQKIH 781YKCHQCGKAFI-------HERTH 782 YKCKECGKAFN-------HHRIH 783YKCKECGQAFR-------HHKLH 784 YKCKQCGKAFG-------HGRTH 785YKCMECGKAFN-------HQRIH 786 YKCPDCGKSFS-------HQRTH 787YMCSECGRGFS-------HQRTH 788 YQCNICGKCFS-------HQRTH 789YRCEECGKAFR-------HKRIH 790 YRCKYCDRSFS-------HVRNIH 791YRCSWEGCEWRFA-------HFRKH 792 YSCGICGKSFS-------HCILH 793YTCKQCGKAFS-------HETTH 794 YTCSDCGKAFR-------HRRTH 795YTCSYCGKSFT-------HTRIH 796 YVCDVEGCTWKFA-------HKKRH 797YVCRECGRGFR-------HKRTH 798 YVCRECRRGFS-------HQRTH 799YVCSKCGKAFT-------HQKIH 800 TGEKPFQCRICMRNFS-------HLRTH 807

An at least 10× zinc finger, or a zinc finger of another length (e.g.,selected for a non-human or other application), may contain the zincfinger modules in which each of the recognition helices is inserted intothe same conserved sequence. In another embodiment, an ablator maycontain zinc finger modules with contain recognition helices insertedinto different conserved sequences. In the examples herein, theconserved sequence of (N-terminus)—PGEKPYKCPECGKSFS-XXXXXXX-HQRTH(carboxy terminus), COOH [SEQ ID NO: 745] was used or(N-terminus)—TGEKPFQCRICMRNFS-XXXXXXX-HLRTH (carboxy terminus)—COOH,wherein XXXXXXX [SEQ ID NO: 807] is the zinc finger recognition helixwhere used. However, the invention is not limited to these sequences.

According to the invention, in one embodiment, the at least 30 nucleicacid sequence may be 32 base pairs in length in order to accommodationalternate reading frames for the zinc finger.

Depending upon the sequence of the unique nucleic acid sequence of theablation site, specific zinc finger helices are selected for insertioninto the zinc finger conserved sequence. In some embodiments, the uniquenucleic acid sequence of the ablation site may contain more than one ofthe same three base pair triplet. In this instance, one may select thesame recognition helix or a different recognition helix.

In one embodiment, the zinc finger for the at least 30 base pairsequence: SEQ ID NO: 806: GGTCGATGTTCGCAACGTCGATCGTACGTGCA wasengineered, so that the zinc finger comprises a nucleic acid sequenceencoding at least ten zinc fingers consisting of: (a) a first N-terminalzinc finger comprising a recognition helix which specifically binds toTGC; (b) second zinc finger comprising a recognition helix whichspecifically binds to ACG; (c) a third zinc finger comprising arecognition helix which specifically binds to CGT; (d) a fourth zincfinger comprising a recognition helix which specifically binds to GAT;(e) a fifth zinc finger comprising a recognition helix whichspecifically binds to GTC; (f) a sixth zinc finger comprising arecognition helix which specifically binds to AAC; (g) a seventh zincfinger comprising a recognition helix which binds to CGC; (h) an eighthzinc finger comprising a recognition helix that specifically binds toGTT; (i) an ninth zinc finger comprising a recognition helix thatspecifically binds to GAT; and (j) a tenth zinc finger comprising arecognition helix which specifically binds to GTC.

In the examples provided herein, the recognition helix of (a) whichspecifically binds to TGC is QRRSLGH (SEQ ID NO: 663); the recognitionhelix of (b) which specifically binds to ACG is KKNDLTR (aa 29-56 of SEQID NO: 60); the recognition helix of (c) which specifically binds to CGTis SRRTCRA (SEQ ID NO: 155); the recognition helix of (d) whichspecifically binds to GAT is VRHNLTR (SEQ ID NO: 270); the recognitionhelix of (e) which specifically binds to GTC is DRTSLAR (SEQ ID NO:540); the recognition helix of (f) which specifically binds to AAC isDSGNLRV (SEQ ID NO: 64); the recognition helix of (g) which specificallybinds to CGC is HTGHLLE (SEQ ID NO: 151); the recognition helix of (h)which specifically binds to GTT is TNQALGV (aa 197-224 of SEQ ID NO:60); the recognition helix of (j) which specifically binds to GAT isVRHNLTR (SEQ ID NO: 270); and the recognition helix of (k) whichspecifically binds to GTC is DRTSLAR (SEQ ID NO: 540). However, otherrecognition helices exist for many of these triplets. For example, therecognition helix of (a) which specifically binds to TGC may be selectedfrom the group consisting of: ARNTLVH, QRRSLGH, QARSLRA, QQRSLKN, andQNRSLAH, QGRSLRA, RARNLTL, RGRNLEM, RKRNLIM, RMRNLII, RNRNLVL, RRRNLHL,RRRNLTL, RSRNLDI, RSRNLLL, and RSRNLTL (SEQ ID NO: 658-673); therecognition helix of (b) which specifically binds to ACG may be selectedform the group consisting of: KNNDLTR; KRIDLQR; RKHDLNM; RRQTLRQ;KGNDLTR; RNITLVR, RSHDLTV, ASADLTR, QNATRKR, QSGDLTR, RSQTLAQ; andRTDTLRD (SEQ ID NO: 104-119); the recognition helix of (c) whichspecifically binds to CGT may be selected from the group consisting ofRSQTRKT (SEQ ID NO: 154) and SRRTCRA (SEQ ID NO: 155); the recognitionhelix of (d) which specifically binds to GAT may be selected from thegroup consisting of VRHNLTR, ISHNLAR, ISSNLQR, LGNNLKR, LNSNLAR,LSTNLTR, LTHNLRR, QSSNLAR, RSDALIQ, SKQALAV, TGQQLRV, TKQRLVV, TRQRLRI,TSANLSR, TSGNLVR, TSQMLVV, TSSNLSR, TTSNLRR, VGHNLSR, VGSNLTR (SEQ IDNO: 251-270); the recognition helix of (e) which specifically binds toGTC may be selected from the group consisting of DRTSLAR, DHSSLKR,APSSLRR, DATQLVR, DPGALVR, DPTSLNR, DRSALAR, DRSALSR, DRSSLRR, DRTPLNR,DRTPLQN, EGGALRR, ESGALRR, NTSLLRR, RSDVLSE, TGAVLRR, TGAVLTR, TKKILTV,TKSLLAR, TMAVLRR, TRAVLRR, TSTILAR, TSTLLKR, and TSTLLNR (SEQ ID NO:530-553); the recognition helix of (f) which specifically binds to AACmay be selected from the group consisting of DRSNRKT, DSGNLRV, GASALRQ,GASALRS, GGTALRM, GGTALVM, GHTALAL, GHTALRH, GHTALRN, GPTALVN, andHRTNLIA (SEQ ID NO: 63-73); the recognition helix of (g) whichspecifically binds to CGC may be HTGHLLE (SEQ ID NO: 151); and therecognition helix of (h) which specifically binds to GTT may be selectedfrom the group consisting of HKSSLTR, TNQALGV, AATALRR, HHNSLTR,HSSSLVR, IKAILTR, INHSLRR, IRTSLKR, MNSVLKR, MTSSLRR, QATLLRR, QSSALTR,THTVLAR, TKPVLKI, TNSVLGR, TRHSLGR, TSGALTR, TSGSLTR, TSGSLVR, TSTLLKR,TSTRLDI, TTALLKR, TTSALTR, TTTVLAR, and VGGSLNR (SEQ ID NO: 583-607);the recognition helix of (i) which specifically binds to GAT may beselected from the group consisting of ISHNLAR, VRHNLTR, ISSNLQR,LGNNLKR, LNSNLAR, LSTNLTR, LTHNLRR, QSSNLAR, RSDALIQ, SKQALAV, TGQQLRV,TKQRLVV, TRQRLRI, TSANLSR, TSGNLVR, TSQMLVV, TSSNLSR, TTSNLRR, VGHNLSR,and VGSNLTR (SEQ ID NO: 251-270); and the recognition helix of (j) whichspecifically binds to GTC may be selected from the group consisting ofDRTSLAR, DHSSLRKR, APSSLRR, DATQLVR, DPGALVR, DPTSLNR, DRSALAR, DRSALSR,DRSSLRR, DRTPLNR, DRTPLQN, EGGALRR, ESGALRR, NTSLLRR, RSDVLSE, TGAVLRR,TGAVLTR, TKKILTV, TKSLLAR, TMAVLRR, TRAVLRR, TSTILAR, TSTLLKR, andTSTLLNR (SEQ ID NO: 530-553).

Still other recognition helices for these and other triplets may beutilized in various embodiments of the invention. The following Table 3provides illustrative recognition helices for various three base pairtriplets.

TABLE 3 Triplet Recognition Helix (SEQ ID NO:) AAA QRANL (SEQ ID NO: 61)QRSNLKV (SEQ ID NO: 62) AAC DRSNRKT (SEQ ID NO: 63)GGTALVM (SEQ ID NO: 68) DSGNLRV (SEQ ID NO: 64) GHTALAL (SEQ ID NO: 69)GASALRQ (SEQ ID NO: 65) GHTALRH (SEQ ID NO: 70) GASALRS (SEQ ID NO: 66)GHTALRN (SEQ ID NO: 71) TALRM (SEQ ID NO: 67) GPTALVN (SEQ ID NO: 72)HRTNLIA (SEQ ID NO: 73) AAG RKDNLKN (SEQ ID NO: 74)RSDNLSV (SEQ ID NO: 76) RSANLSV (SEQ ID NO: 75) RSDNLTQ (SEQ ID NO: 77)AAT TSSNRKT (SEQ ID NO: 78) VSSNLNV (SEQ ID NO: 80)TTGNLTV (SEQ ID NO: 79) ACA QNATRIN (SEQ ID NO: 81)SPADLTR (SEQ ID NO: 82) ACC DKKDLTR (SEQ ID NO: 83) ACGASADLTR (SEQ ID NO: 84) RKHDLNM (SEQ ID NO: 90) KGNDLTR (SEQ ID NO: 85)RNITLVR (SEQ ID NO: 91) KNNDLTR (SEQ ID NO: 86) RRQTLRQ (SEQ ID NO: 92)KRIDLQR (SEQ ID NO: 87) RSHDLTV (SEQ ID NO: 93) QNATRKR (SEQ ID NO: 88)RSQTLAQ (SEQ ID NO: 94) QSGDLTR (SEQ ID NO: 89) RTDTLRD (SEQ ID NO: 95)ACT ARSTRTN (SEQ ID NO: 96) THLDLIR (SEQ ID NO: 98)HASTRHC (SEQ ID NO: 97) AGA KNWKLQA (SEQ ID NO: 99)QSSHLTT (SEQ ID NO: 101) QLAHLRA (SEQ ID NO: 100)RSANLAR (SEQ ID NO: 102) AGC ERSHLRE (SEQ ID NO: 103) AGGDSAHLTR (SEQ ID NO: 104) RRAHLRQ (SEQ ID NO: 112)QSAHRTK (SEQ ID NO: 105) RRTHLRV (SEQ ID NO: 113)RGNHLVV (SEQ ID NO: 106) RSDHLKT (SEQ ID NO: 114)RMAHLHA (SEQ ID NO: 107) RSDHLSA (SEQ ID NO: 115)RNEHLKV (SEQ ID NO: 108) RSDHLSQ (SEQ ID NO: 116)RPHHLDA (SEQ ID NO: 109) RSDHLTN (SEQ ID NO: 117)RRAHLLN (SEQ ID NO: 110) RSDHLTQ (SEQ ID NO: 118)RRAHLLS (SEQ ID NO: 111) RSSHLKM (SEQ ID NO: 119) AGTHRTTLRTN (SEQ ID NO: 120) QSAHLST (SEQ ID NO: 121) ATAQKSSLIA (SEQ ID NO: 122) ATG RRDELNV (SEQ ID NO: 123)RSDSLSV (SEQ ID NO: 124) ATT HKNALQN (SEQ ID NO: 125) CAADRANLST (SEQ ID NO: 126) QSGNLTE (SEQ ID NO: 128)QKSNLII (SEQ ID NO: 127) QSSNLTV (SEQ ID NO: 129) CACSKKALTE (SEQ ID NO: 130) CAG DSANRTK (SEQ ID NO: 131)RSDNLSE (SEQ ID NO: 134) RADNLTE (SEQ ID NO: 132)RTDYLVD (SEQ ID NO: 135) RSDNLRE (SEQ ID NO: 133) CATDRSNRIK (SEQ ID NO: 136) TSGNLTE (SEQ ID NO: 137) CCADRSDLSR (SEQ ID NO: 138) QNSTRIG (SEQ ID NO: 140)NRTDLIR (SEQ ID NO: 139) TSHSLTE (SEQ ID NO: 141) CCCSKKHLAE (SEQ ID NO: 142) CCG DSSSLTR (SEQ ID NO: 143)RNDTLTE (SEQ ID NO: 145) DYDVRKR (SEQ ID NO: 144)RSDTLSE (SEQ ID NO: 146) CCT TKNSLTE (SEQ ID NO: 147 CGAQSGHLTE (SEQ ID NO: 148) QSTHLTQ (SEQ ID NO: 150)QSSHLNV (SEQ ID NO: 149) CGC HTGHLLE (SEQ ID NO: 151) CGGRSDNLTE (SEQ ID NO: 152) RSDKLTE (SEQ ID NO: 153) CGTRSQTRKT (SEQ ID NO: 154) SRRTCRA (SEQ ID NO: 155) CTADSSSRTK (SEQ ID NO: 156) QNSTLTE (SEQ ID NO: 157) CTCASDDLTQ (SEQ ID NO: 158) CTG HNYARDC (SEQ ID NO: 159)RSDALSA (SEQ ID NO: 162) RNDALTE (SEQ ID NO: 160)RSDALSN (SEQ ID NO: 163) RSDALRE (SEQ ID NO: 161)RSDTLSE (SEQ ID NO: 164) CTT TTGALTE (SEQ ID NO: 165) GAAHKPNLHR (SEQ ID NO: 166) QRNNLGR (SEQ ID NO: 181)HRPNLTR(SEQ ID NO: 167) QRSNLAR(SEQ ID NO: 182) LGENLRR (SEQ ID NO: 168)QRSNLVR (SEQ ID NO: 183) QASNLAR(SEQ ID NO: 169) QRTNLQR(SEQ ID NO: 184)QASNLLR(SEQ ID NO: 170) QSGNLAR (SEQ ID NO: 185) QASNLTR(SEQ ID NO: 171)QSNNLNR (SEQ ID NO: 186) QDGNLGR(SEQ ID NO: 172) QSSNLTK(SEQ ID NO: 187)QDGNLTR(SEQ ID NO: 173) QSSNLTR(SEQ ID NO: 188) QGSNLAR(SEQ ID NO: 174)QSSNLVR(SEQ ID NO: 189) QHPNLTR(SEQ ID NO: 175) QTNNLGR(SEQ ID NO: 190)QKGNLLR(SEQ ID NO: 176) QTNNLNR(SEQ ID NO: 191) QKSNLIR(SEQ ID NO: 177)QTNNLTR(SEQ ID NO: 192) QLSNLTR(SEQ ID NO: 178) QTVNLDR(SEQ ID NO: 193)QQSNLSR (SEQ ID NO: 179) RKPNLLR(SEQ ID NO: 194) QQTNLTR(SEQ ID NO: 180)TTTNLRR(SEQ ID NO: 195) GAC CPSNLRR (SEQ ID NO: 196)DRANLSR(SEQ ID NO: 207) DDANLRR(SEQ ID NO: 197) DRGNLTR(SEQ ID NO: 208)DEANLRR (SEQ ID NO: 198) DRSNLTR(SEQ ID NO: 209) DLSNLKR(SEQ ID NO: 199)EEANLRR(SEQ ID NO: 210) DMGNLGR(SEQ ID NO: 200) EESNLRR(SEQ ID NO: 211)DPANLRR(SEQ ID NO: 201) EEVNLRR(SEQ ID NO: 212) DPGNLVR(SEQ ID NO: 202)EGGNLMR(SEQ ID NO: 213) DPSNLIR(SEQ ID NO: 203) EKANLTR(SEQ ID NO: 214)DPSNLQR(SEQ ID NO: 204) EQANLRR(SEQ ID NO: 215) DPSNLRR(SEQ ID NO: 205)HSSNFNK(SEQ ID NO: 216) DQGNLIR(SEQ ID NO: 206) RSDNLSE(SEQ ID NO: 217)GAG KHSNLAR(SEQ ID NO: 218) RQMNLDR(SEQ ID NO: 234)KHSNLTR(SEQ ID NO: 219) RRDNLLR(SEQ ID NO: 235) KKTNLTR(SEQ ID NO: 220)RRDNLNR(SEQ ID NO: 236) KSSNLRR(SEQ ID NO: 221) RSANLTR(SEQ ID NO: 237)QSFNLRR(SEQ ID NO: 222) RSDHLSR(SEQ ID NO: 238) REDNLGR(SEQ ID NO: 223)RSDNLAR(SEQ ID NO: 239) RGDNLKR(SEQ ID NO: 224) RSDNLSR(SEQ ID NO: 240)RGDNLNR(SEQ ID NO: 225) RSDNLST (SEQ ID NO: 241) RHDQLTR(SEQ ID NO: 226)RSDNLTR (SEQ ID NO: 242) RIDNLGR(SEQ ID NO: 227)RSDNLVR (SEQ ID NO: 243) RKSNLIR(SEQ ID NO: 228) RSSNLQR(SEQ ID NO: 244)RMSNLDR(SEQ ID NO: 229) RTHNLKR(SEQ ID NO: 245) RNTNLTR(SEQ ID NO: 230)RTHNLTR(SEQ ID NO: 246) RPHNLLR(SEQ ID NO: 231) RVDNLPR(SEQ ID NO: 247)RQDNLGR(SEQ ID NO: 232) SGSNFTR(SEQ ID NO: 248) RQDNLQR(SEQ ID NO: 233)TNNNLAR(SEQ ID NO: 249) VHWNLMR(SEQ ID NO: 250) GATISHNLAR (SEQ ID NO: 251) TKQRLVV(SEQ ID NO: 261) ISSNLQR(SEQ ID NO: 252)TRQRLRI(SEQ ID NO: 262) LGNNLKR(SEQ ID NO: 253) TSANLSR(SEQ ID NO: 263)LNSNLAR(SEQ ID NO: 254) TSGNLVR(SEQ ID NO: 264) LSTNLTR(SEQ ID NO: 255)TSQMLVV(SEQ ID NO: 265) LTHNLRR(SEQ ID NO: 256) TSSNLSR(SEQ ID NO: 266)QSSNLAR(SEQ ID NO: 257) TTSNLRR(SEQ ID NO: 267) RSDALIQ(SEQ ID NO: 238)VGHNLSR(SEQ ID NO: 268) SKQALAV(SEQ ID NO: 259) VGSNLTR(SEQ ID NO: 269)TGQQLRV(SEQ ID NO: 260) VRHNLTR(SEQ ID NO: 270) GCADKAQLGR(SEQ ID NO: 271) QPNTLTR(SEQ ID NO: 286) DRSALSR(SEQ ID NO: 272)QRGTLNR(SEQ ID NO: 287) DRSQLAR(SEQ ID NO: 273) QSGDLRR(SEQ ID NO: 288)ERGTLAR(SEQ ID NO: 274) QSGDLTR(SEQ ID NO: 289) HNGTLKR(SEQ ID NO: 275)QSGSLTR(SEQ ID NO: 290) KNTRLSV(SEQ ID NO: 276) QSNVLSR(SEQ ID NO: 291)LKHSLLR(SEQ ID NO: 277) QSTTLKR(SEQ ID NO: 292) LNHTLKR(SEQ ID NO: 278)QTATLKR(SEQ ID NO: 293) LRHSLSR(SEQ ID NO: 279) QTNTLKR(SEQ ID NO: 294)QDNTLRR(SEQ ID NO: 280) RGQELRR(SEQ ID NO: 295) QDVSLVR(SEQ ID NO: 281)RRQELHR(SEQ ID NO: 296) QGGTLRR(SEQ ID NO: 282) RRQELKR(SEQ ID NO: 297)QGNTLTR(SEQ ID NO: 283) RRQELTR(SEQ ID NO: 298) QKGTLGR(SEQ ID NO: 284)RRVDLLR(SEQ ID NO: 299) QNGTLTR(SEQ ID NO: 285) SPEQLAR(SEQ ID NO: 300)GCC DCRDLAR(SEQ ID NO: 301) DSPTLRR(SEQ ID NO: 313)DGSTLNR(SEQ ID NO: 302) DSSVLRR(SEQ ID NO: 314) DGSTLRR(SEQ ID NO: 303)EHRGLKR(SEQ ID NO: 315) DHSNLSR(SEQ ID NO: 304) ERGTLAR(SEQ ID NO: 316)DKSCLNR(SEQ ID NO: 305) ERRGLAR(SEQ ID NO: 317) DKSVLAR(SEQ ID NO: 306)ERRGLDR(SEQ ID NO: 318) DPSNLRR(SEQ ID NO: 307) KRRDLDR(SEQ ID NO: 319)DPSTLRR(SEQ ID NO: 308) LKKDLLR(SEQ ID NO: 320) DRRTLDR(SEQ ID NO: 309)SHTVLTR(SEQ ID NO: 321) DRSDLTR(SEQ ID NO: 310) SKKSLTR(SEQ ID NO: 322)DRSSLTR(SEQ ID NO: 311) SNKDLTR(SEQ ID NO: 323) DRSSRTK(SEQ ID NO: 312)VRKDLTR(SEQ ID NO: 324) GCG KADTLVR(SEQ ID NO: 325)RRHTLTR(SEQ ID NO: 343) KHDTLHR(SEQ ID NO: 326) RRLTLLR(SEQ ID NO: 344)KNNDLTR(SEQ ID NO: 327) RSDDLQR(SEQ ID NO: 345) RADTLRR(SEQ ID NO: 328)RSDDLTR(SEQ ID NO: 346) RAHTLRR(S£Q ID NO: 329) RSDDLVR(SEQ ID NO: 347)REDSLPR(SEQ ID NO: 330) RSDELNR(SEQ ID NO: 348) RHAALLS(SEQ ID NO: 331)RSDELQR(SEQ ID NO: 349) RKDGLTR(SEQ ID NO: 332) RSDELSR(SEQ ID NO: 350)RKGTLDR(SEQ ID NO: 333) RSDELTR(SEQ ID NO: 351) RKLGLLR(SEQ ID NO: 334)RSDERKR(SEQ ID NO: 352) RLDMLAR(SEQ ID NO: 335) RSDSLSK(SEQ ID NO: 353)RLRDLPR(SEQ ID NO: 336) RSDTLKK(SEQ ID NO: 354) RNLTLAR(SEQ ID NO: 337)RSDVLTR(SEQ ID NO: 355) RNLTLVR(SEQ ID NO: 338) RSNTLLR(SEQ ID NO: 356)RPDGLAR(SEQ ID NO: 339) RTDLLRR(SEQ ID NO: 357) RRDDLTR(SEQ ID NO: 340)RTDSLPR(SEQ ID NO: 358) RRDGLTR(SEQ ID NO: 341) RTDTLAR(SEQ ID NO: 359)RRHGLDR(SEQ ID NO: 342) RVDDLGR(SEQ ID NO: 360) GCTARSTRTT(SEQ ID NO: 361) QSSDLRR(SEQ ID NO: 377) EGSGLKR{SEQ ID NO: 362)QSSDLSR(SEQ ID NO: 378) GATALKR(SEQ ID NO: 363) QSSDLTR(SEQ ID NO: 379)KHQTLQR(SEQ ID NO: 364) THSMLAR(SEQ ID NO: 380) LKHDLRR(SEQ ID NO: 365)TKPILVR(SEQ ID NO: 381) LRASLRR(SEQ ID NO: 366) TKQILGR(SEQ ID NO: 382)LRQTLAR(SEQ ID NO: 367) TKQVLDR(SEQ ID NO: 383) LRTSLVR(SEQ ID NO: 368)TSGELVR(SEQ ID NO: 384) MKNTLTR(SEQ ID NO: 369) TSSGLTR(SEQ ID NO: 385)NGQGLRR(SEQ ID NO: 370) TTQALRR(SEQ ID NO: 386) NKQALDR(SEQ ID NO: 371)VGASLKR(SEQ ID NO: 387) NRSDRTR(SEQ ID NO: 372) VGNSLTR(SEQ ID NO: 388)QRQALDR(SEQ ID NO: 373) VKNTLTR(SEQ ID NO: 389) QRSDLHR(SEQ ID NO: 374)VRQGLTR(SEQ ID NO: 390) QRSDLTR(SEQ ID NO: 375) VSNSLAR(SEQ ID NO: 391)QSSDLQR(SEQ ID NO: 376) VSNTLTR(SEQ ID NO: 392) GGADKTKLNV(SEQ ID NO: 393) QSQHLVR(SEQ ID NO: 409) DKTKLRV(SEQ ID NO: 394)QSTHLTR(SEQ ID NO: 410) DNAHLAR(SEQ ID NO: 395) QTTHLRR(SEQ ID NO: 411)QANHLSR(SEQ ID NO: 396) QTTHLSR(SEQ ID NO: 412) QGGHLKR(SEQ ID NO: 397)QTTHLSR(SEQ ID NO: 413) QHSHLVR(SEQ ID NO: 398) QVSHLTR(SEQ ID NO: 414)QKPHLSR(SEQ ID NO: 399) RMERLDR(SEQ ID NO: 415) QMSHLKR(SEQ ID NO: 400)RPAKLVL(SEQ ID NO: 416) QNSHLRR(SEQ ID NO: 401) RPSKLVL(SEQ ID NO: 417)QNSHLRR(SEQ ID NO: 402) RRDHRTH(SEQ ID NO: 418) QRAHLER(SEQ ID NO: 403)RSTHLRV(SEQ ID NO: 419) QRAHLIR(SEQ ID NO: 404) RTDRLIR(SEQ ID NO: 420)QSAHLKR(SEQ ID NO: 405) THAHLTR(SEQ ID NO: 421) QSGHLAR(SEQ ID NO: 406)TSAHLAR(SEQ ID NO: 422) QSGHLQR(SEQ ID NO: 407) YNTHLTR(SEQ ID NO: 423)QSGHLSR(SEQ ID NO: 408) GGC AKSKLDR(SEQ ID NO: 424)KNHSLNN(SEQ ID NO: 437) APSKLDR(SEQ ID NO: 425) KNVSLTH(SEQ ID NO: 438)DGGHLTR(SEQ ID NO: 426) LKEHLTR(SEQ ID NO: 439) DKSHLPR(SEQ ID NO: 427)QSSHLAR(SEQ ID NO: 440) DPGHLVR(SEQ ID NO: 428) SKHKLER(SEQ ID NO: 441)DRSHLAR(SEQ ID NO: 429) SPSKLAR(SEQ ID NO: 442) DRSHLSR(SEQ ID NO: 430)SPSKLVR(SEQ ID NO: 443) DRSHLTR(SEQ ID NO: 431) TNSKLTR(SEQ ID NO: 444)EKSHLKR(SEQ ID NO: 432) TPSKLDR(SEQ ID NO: 445) EKSHLTR(SEQ ID NO: 433)TRAKLHK(SEQ ID NO: 446) ENSKLNR(SEQ ID NO: 434) VPSKLAR(SEQ ID NO: 447)ESGHLKR(SEQ ID NO: 435) VPSKLKR(SEQ ID NO: 448) ESGHLRR(SEQ ID NO: 436)VPSKLLR(SEQ ID NO: 449) GGG KGDHLRR(SEQ ID NO: 450)RNTHLAR(SEQ ID NO: 464) KKDHLHR(SEQ ID NO: 451) RQGHLKR(SEQ ID NO: 465)KRERLDR(SEQ ID NO: 452) RRAHLQN(SEQ ID NO: 466) KRERLER(SEQ ID NO: 453)RREHLVR(SEQ ID NO: 467) KSNHLHV(SEQ ID NO: 454) RRSHLTR(SEQ ID NO: 468)KTSHLRA(SEQ ID NO: 455) RSAHLAR(SEQ ID NO: 469) RGDKLAL(SEQ ID NO: 456)RSAHLSR(SEQ ID NO: 470) RGDKLGP(SEQ ID NO: 457) RSDHLAR(SEQ ID NO: 471)RGNHLRR(SEQ ID NO: 458) RSDHLSK(SEQ ID NO: 472) RIDKLGG(SEQ ID NO: 459)RSDHLSR(SEQ ID NO: 473) RKHHLGR(SEQ ID NO: 460) RSDHLTR(SEQ ID NO: 474)RKHRLDG(SEQ ID NO: 461) RSDKLNR(SEQ ID NO: 475) RNDKLVP(SEQ ID NO: 462)RSDKLVR(SEQ ID NO: 476) RNHGLVR(SEQ ID NO: 463) RTEHLAR(SEQ ID NO: 477)GGT DSSKLSR(SEQ ID NO: 478) RRQKLTI(SEQ ID NO: 493)EAHHLSR(SEQ ID NO: 479) RRSRLVR(SEQ ID NO: 494) HGHRLKT(SEQ ID NO: 480)RSDHLST(SEQ ID NO: 495) IPNHLAR(SEQ ID NO: 481) RSDHLTT(SEQ ID NO: 496)IRHHLKR(SEQ ID NO: 482) TKQKLQT(SEQ ID NO: 497) LTQGLRR(SEQ ID NO: 483)TKQRLEV(SEQ ID NO: 498) MGHHLKR(SEQ ID NO: 484) TRQKLET(SEQ ID NO: 499)MKHHLAR(SEQ ID NO: 485) TRTRLVI(SEQ ID NO: 500) MKHHLDA(SEQ ID NO: 486)TSGHLSR(SEQ ID NO: 501) MSDHLSR(SEQ ID NO: 487) TSGHLVR(SEQ ID NO: 502)MSHHLSR(SEQ ID NO: 488) TTTKLAI(SEQ ID NO: 502) QPHHLPR(SEQ ID NO: 489)VDHHLRR(SEQ ID NO: 504) QSSHLAR (SEQ ID NO: 490) VKHGLGR(SEQ ID NO: 505)QSSHLTR(SEQ ID NO: 491) VKHGLTR(SEQ ID NO: 506) RQSRLQR(SEQ ID NO: 492)WPSNLTR(SEQ ID NO: 507) YNWHLQR(SEQ ID NO: 508) GTAQGGALQR (SEQ ID NO: 509) QRSSLVR(SEQ ID NO: 520) QGTSLAR(SEQ ID NO: 510)QSGALAR(SEQ ID NO: 521) QKQALDR(SEQ ID NO: 511) QSGALTR(SEQ ID NO: 522)QKQALTR(SEQ ID NO: 512) QSGSLTR(SEQ ID NO: 523) QKVSLKR(SEQ ID NO: 513)QSGTLTR(SEQ ID NO: 524) QMNALQR(SEQ ID NO: 514) QSSSLIR(SEQ ID NO: 525)QQQALKR(SEQ ID NO: 515) OSSSLVR(SEQ ID NO: 526) QQQALTR(SEQ ID NO: 516)QSSTLTR(SEQ ID NO: 527) QQQALVR(SEQ ID NO: 517) QSTSLQR(SEQ ID NO: 528)QQSSLLR(SEQ ID NO: 518) TSSARTT(SEQ ID NO: 529) QRASLTR(SEQ ID NO: 529)GTC APSSLRR(SEQ ID NO: 530) ESGALRR(SEQ ID NO: 542)DATQLVR(SEQ ID NO: 531) NTSLLRR(SEQ ID NO: 543) DHSSLKR(SEQ ID NO: 532)RSDVLSE(SEQ ID NO: 544) DPGALVR(SEQ ID NO: 533) TGAVLRR(SEQ ID NO: 545)DPTSLNR(SEQ ID NO: 534) TGAVLTR(SEQ ID NO: 546) DRSALAR(SEQ ID NO: 535)TKKILTV(SEQ ID NO: 547) DRSALSR(SEQ ID NO: 536) TKSLLAR(SEQ ID NO: 548)DRSSLRR{SEQ ID NO: 537) TMAVLRR(SEQ ID NO: 549) DRTPLNR(SEQ ID NO: 538)TRAVLRR(SEQ ID NO: 550) DRTPLQN(SEQ ID NO: 539) TSTILAR(SEQ ID NO: 551)DRTSLAR(SEQ ID NO: 540) TSTLLKR(SEQ ID NO: 552) EGGALRR(SEQ ID NO: 541)TSTLLNR(SEQ ID NO: 553) GTG RASVLDI (SEQ ID NO: 554)RRFILSR(SEQ ID NO: 569) RGDALAR(SEQ ID NO: 555) RRHILDR(SEQ ID NO: 570)RHTSLTR(SEQ ID NO: 556) RSAALSR(SEQ ID NO: 571) RKDALHV(SEQ ID NO: 557)RSDALAR(SEQ ID NO: 572) RKHILIH(SEQ ID NO: 558) RSDALRT(SEQ ID NO: 573)RKTALNR(SEQ ID NO: 559) RSDALSR(SEQ ID NO: 574) RNFILAR(SEQ ID NO: 560)RSDALTR(SEQ ID NO: 575) RNFILQR(SEQ ID NO: 561) RSDELVR(SEQ ID NO: 576)RNHVLAR(SEQ ID NO: 562) RSHILTN(SEQ ID NO: 577) RNTALQH(SEQ ID NO: 563)RTSSLKR(SEQ ID NO: 578) RNVALGN(SEQ ID NO: 564) RTVALNR(SEQ ID NO: 579)RNVNLVT(SEQ ID NO: 565) SRFTLGR(SEQ ID NO: 580) RPDALPR(SEQ ID NO: 566)SRFTLGR(SEQ ID NO: 581) RRAALGP(SEQ ID NO: 567) VSSSLRR(SEQ ID NO: 582)RREVLEN(SEQ ID NO: 568) GTT AATALRR(SEQ ID NO: 583)TKPVLKK(SEQ ID NO: 595) HHNSLTR(SEQ ID NO: 584) TRHSLGR(SEQ ID NO: 598)HKSSLTR(SEQ ID NO: 585) TSGALTR(SEQ ID NO: 599) HSSSLVR(SEQ ID NO: 586)TSGSLTR(SEQ ID NO: 600) IKAILTR(SEQ ID NO: 587) TSGSLVR(SEQ ID NO: 601)INHSLRR(SEQ ID NO: 588) TSTLLKR(SEQ ID NO: 602) IRTSLKR(SEQ ID NO: 589)TSTRLDK(SEQ ID NO: 603) MNSVLKR (SEQ ID NO: 590) TTALLKR(SEQ ID NO: 604)MTSSLRR(SEQ ID NO: 591) TTSALTR(SEQ ID NO: 605) QATLLRR(SEQ ID NO: 592)TTTVLAR(SEQ ID NO: 606) QSSALTR(SEQ ID NO: 593) VGGSLNR(SEQ ID NO: 607)THTVLAR(SEQ ID NO: 594) TAA QGGNLAL(SEQ ID NO: 608)QQGNLRN(SEQ ID NO: 611) QGGNLTL(SEQ ID NO: 609) QRGNLNM(SEQ ID NO: 612)QQGNLQL (SEQ ID NO: 610) QSGNLHT(SEQ ID NO: 613) TACNSDHLTN (SEQ ID NO: 614) TAG QGYNLAG (SEQ ID NO: 615)RPESLAP (SEQ ID NO: 625) RAHNLLL (SEQ ID NO: 616)RPESLRP (SEQ ID NO: 626) REDNLHT (SEQ ID NO: 617)RRDGLAG (SEQ ID NO: 627) RGHNLLV (SEQ ID NO: 618)RRDHLSL (SEQ ID NO: 628) RGTNLRT(SEQ ID NO: 619)RRDHLSP (SEQ ID NO: 629) RHDGLAG(SEQ ID NO: 620)RRDNLPK (SEQ ID NO: 630) RIDHLVP(SEQ ID NO: 621)RRRNLQI (SEQ ID NO: 631) RKTGLLI(SEQ ID NO: 622)RSHNLKL (SEQ ID NO: 632) RLDGLAG (SEQ ID NO: 623)RSHNLRL (SEQ ID NO: 633) RPEGLST(SEQ ID NO: 624)TSSNRKK (SEQ ID NO: 634) TCA QSADRTK (SEQ ID NO: 635) TCCDKRSLPH(SEQ ID NO: 636) TCG ASSTRTK (SEQ ID NO: 637)RMDSLGG (SEQ ID NO: 644) HSSDLTR (SEQ ID NO: 638)RRDGLSG (SEQ ID NO: 645) KNNDLLK (SEQ ID NO: 639)RSDELRT (SEQ ID NO: 646) NRSDLSR (SEQ ID NO: 640)RSDGLRG (SEQ ID NO: 647) QSSDLSK (SEQ ID NO: 641)RSDTLPA (SEQ ID NO: 648) RADGLQL (SEQ ID NO: 642)RSDTLPL (SEQ ID NO: 649) RGDSLKK (SEQ ID NO: 643)RSSDLSR (SEQ ID NO: 650) RTDSLQP (SEQ ID NO: 651) TCTNNRDRTK (SEQ ID NO: 652) SKPNLKM (SEQ ID NO: 654)QRNTLKG (SEQ ID NO: 653) TGA QAGHLAS(SEQ ID NO: 655)QSGHLTK (SEQ ID NO: 657) QREHLTT (SEQ ID NO: 656) TGCANRTLVH (SEQ ID NO: 658) RRRNLHL (SEQ ID NO: 669)QARSLRA (SEQ ID NO: 659) RRRNLTL (SEQ ID NO: 670)QGRSLRA (SEQ ID NO: 660) RSRNLDI (SEQ ID NO: 671)QNRSLAH (SEQ ID NO: 661) RSRNLLL (SEQ ID NO: 672)QQRSLKN (SEQ ID NO: 662) RSRNLTL (SEQ ID NO: 673)QRRSLGH (SEQ ID NO: 663) RARNLTL (SEQ ID NO: 664)RGRNLEM (SEQ ID NO: 665) RKRNLIM (SEQ ID NO: 666)RMRNLII (SEQ ID NO: 667) RNRNLVL (SEQ ID NO: 668) TGGRMDHLAG (SEQ ID NO: 674) RSDHLSL (SEQ ID NO: 679)RNAHRIN (SEQ ID NO: 675) RSDHLST (SEQ ID NO: 680)RRDHLSL (SEQ ID NO: 676) RSDHLTT (SEQ ID NO: 681)RREHLTI (SEQ ID NO: 677) RTESLHI (SEQ ID NO: 682)RSDHLRE (SEQ ID NO: 678) TGT KRQHLEY (SEQ ID NO: 683)QQHGLRH (SEQ ID NO: 691) QAHGLTA (SEQ ID NO: 684)QRHGLSS (SEQ ID NO: 692) QAHGLTG (SEQ ID NO: 685)RHQHLKL (SEQ ID NO: 693) QPGHLTA (SEQ ID NO: 686)RKQHLQL (SEQ ID NO: 694) QPHGLAH (SEQ ID NO: 687)RKQHLTL (SEQ ID NO: 695) QPHGLGA (SEQ ID NO: 688)RKQHLVL (SEQ ID NO: 696) QPHGLRA (SEQ ID NO: 689)RRQALEY (SEQ ID NO: 697) QPHGLRH (SEQ ID NO: 690)RRQHLQY (SEQ ID NO: 698) TTA QQTGLNV (SEQ ID NO: 699) TTCQRNALRG (SEQ ID NO: 700) RANHLTI (SEQ ID NO: 701) TTGRADALMV (SEQ ID NO: 702) RSDSLSA (SEQ ID NO: 703) TTTHSNARKT (SEQ ID NO: 704) QRNAKSG(SEQ ID NO: 705)

In one embodiment, the endonuclease catalytic domain is linked to a zincfinger in which the at least 10 independently selected recognitionhelices are all different. In certain embodiments, it will be desirableto include different recognition helices to a repeated triplet of basepairs in the ablation site. In other embodiments, recognition helices toany repeated triplets are the same. For example, 0, 1, 2, or 3 of therecognition helices selected are the same.

In certain embodiments, an AAV vector contains two or more endonucleaseablation sites, which may the same or different from one another. Theseendonuclease ablation sites may be engineered into the vector in avariety of configurations. More particularly, an AAV vector carrying agene of interest (GOI) is engineered to contain at least one ablationsite 5′ to the coding strand of the gene of interest. Where the vectorcontains two or more ablation sites, the second ablation site may belocated: 3′ to the gene of interest in sense orientation; 3′ to the geneof interest in inverted orientation; two ablation sites can be located5′ to the gene of interest, with one of sense orientation and the otherin inverted orientation, or both in sense orientation; or the vector maycontain one or two ablation sites 5′ to the gene of interest (one insense and one in inverted orientation or both in sense orientation) andtwo ablation sites 3′ to the gene of interest (one in sense and one ininverted orientation or both in sense orientation).

Where two ablation sites are located such that there is no interveninggene of interest, i.e., two 5′ or two 3′ ablation sites, the ablationsites may be separated by a spacer. Any suitable spacer may be selectedand the spacer sequence may be a non-coding sequence. In anotherembodiment, the spacer sequence may be a reporter gene, transgene, orgene of interest.

In one embodiment, when a composition of the invention contains morethan one ablation site, the ablation sites are the same. In anotherembodiment, when a composition or vector system of the inventioncontains more than one ablation site, each site differs from the otherand each is specifically and selectively targeted by a differentchimeric endonuclease.

In the working examples below, an ablator encoded by the sequence:10xZF-FokI_Cat nucleotide sequence: SEQ ID NO: 59, is illustrated:

ATGGGCGAGAAGCCCTACAAGTGCCCTGAGTGCGGCAAGAGCTTCAGCCAGAGAAGAAGCCTGGGCCACCACCAGCGTACGCACCCCGGCGAGAAACCTTATAAGTGTCCCGAATGTGGCAAGTCCTTCAGCAAGAAGAACGACCTGACCCGGCACCAGCGGACACACCCCGGGGAAAAGCCATACAAATGTCCAGAGTGTGGGAAGTCTTTCTCCAGCCGGCGGACCTGCAGAGCCCATCAGAGAACACATACCGGGGAGAAGCCTTTCCAGTGCCGGATCTGCATGAGAAACTTCAGCGTGCGGCACAACCTGACCAGACACCTGAGGACCCATACCGGCGAAAAACCCTTTCAGTGCAGAATCTGTATGCGGAACTTCTCCGACCGGACCAGCCTGGCCCGGCATCTGAGAACTCATCCTGGGGAAAAGCCCTATAAGTGTCCAGAATGCGGGAAATCCTTTAGCGACAGCGGCAACCTGCGGGTGCACCAGAGGACTCATCCAGGCGAGAAACCCTACAAATGCCCCGAATGCGGAAAGTCATTCTCCCACACCGGCCATCTGCTCGAGCATCAGCGGACCCACACTGGGGAGAAACCATTTCAGTGTCGCATCTGTATGAGGAATTTCAGCACCAACCAGGCCCTGGGCGTGCACCTGAGAACACACCCAGGCGAGAAGCCTTACAAGTGTCCAGAGTGCGGAAAGTCATTTTCCGTGCGCCACAATCTGACACGGCATCAGCGCACCCATCCCGGCGAGAAGCCTTACAAATGCCCCGAGTGTGGCAAATCTTTCAGTGACCGGACCTCTCTGGCCAGACATCAGAGGACACACGGCACTAGTGGCAAGCAGCTGGTGAAAAGCGAGCTGGAAGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAATACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACCCCACCCAGGACAGAATCCTGGAAATGAAGGTCATGGAATTTTTCATGAAGGTGTACGGCTACCGGGGCGAGCACCTGGGCGGCAGCAGAAAACCCGACGGCGCCATCTACACCGTGGGCAGCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACAGCGGCGGCTACAACCTGCCCATCGGACAGGCCGACGAGATGCAGAGATACGTGGAAGAGAACCAGACCCGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCCGGCTGAACCACATCACCAACTGCAACGGCGCTGTGCTGAGCGTGGAAGAACTGCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAAGAAGTGCGGCGGAAGTTCAACAACGGCGAGATCAACTTCTGATAG.

The transcribed illustrative ablator has the sequence: 10xZF-FokICatamino acid sequence: SEQ ID NO: 60:

MGEKPYKCPECGKSFSQRRSLGHHQRTHPGEKPYKCPECGKSFSKKNDLTRHQRTHPGEKPYKCPECGKSFSSRRTCRAHQRTHTGEKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDRTSLARHLRTHPGEKPYKCPECGKSFSDSGNLRVHQRTHPGEKPYKCPECGKSFSHTGHLLEHQRTHTGEKPFQCRICMRNFSTNQALGVHLRTHPGEKPYKCPECGKSFSVRHNLTRHQRTHPGEKPYKCPECGKSFSDRTSLARHQRTHGTSGKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF.

In an additional embodiment, the ablator is engineered such that it iscontrolled by a cassette that is activated by a transcription factorfollowing being dimerized by a pharmacologic agent, such as described indetail in this specification and incorporated herein by reference.Typically, this cassette is comprises two transcription units. One ofthe two transcription units encodes the DNA binding domain of thetranscription factor fused to a binding domain for the pharmacologicalagent in operative association with a first promoter; and a second ofthe two transcription units encoding the activation domain of thetranscription factor fused to a binding domain for the pharmacologicalagent in operative association with a second promoter. As described inmore detail elsewhere in the specification and incorporated herein, thefirst promoter and the second promoter are independently selected from aconstitutive promoter and an inducible promoter. In one embodiment, thefirst and second promoters are both constitutive promoters and thepharmacological agent is a dimerizer that dimerizes the domains of thetranscription factor. In another embodiment, one of the first promoterand the second promoters is an inducible promoter. Optionally, the twotranscription units are a bicistronic unit containing an IRES orfurin-2A. Various genetic elements and methods suitable for vectorconstruction are described elsewhere in the specification andincorporated herein by reference.

In a further embodiment, the promoter is controlled by arapamycin—regulatable system and the pharmacological agent is rapamycinor a rapalog.

This embodiment, using the unique (at least 30 bp) nucleic acid sequencein the ablation recognition site and the chimeric endonucleasecontaining at least 10× zinc fingers, can be generated as describedherein and other techniques known to those of skill in the art.

Methods for Generating The Replication-Defective Virus Compositions

Many methods have been established for the efficient production ofrecombinant AAVs (rAAVs) that package a transgene—these can be used oradapted to generate the replication-defective virus compositions of theinvention. In a one system, a producer cell line is transientlytransfected with a construct that encodes the transgene flanked by ITRsand a construct(s) that encodes rep and cap. In a second system, apackaging cell line that stably supplies rep and cap is transientlytransfected with a construct encoding the transgene flanked by ITRs. Ina third system, a stable cell line that supplies the transgene flankedby ITRs and rep/cap is used. One method for minimizing the possibilityof generating replication competent AAV (rcAAV) using these systems isby eliminating regions of homology between regions flanking the rep/capcassette and the ITRs that flank the transgene. However, in each ofthese systems, AAV virions are produced in response to infection withhelper adenovirus or herpesvirus, requiring the separation of the rAAVsfrom contaminating virus.

More recently, systems have been developed that do not require infectionwith helper virus to recover the AAV—the required helper functions(i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, andUL29, and herpesvirus polymerase) are also supplied, in trans, by thesystem. In these newer systems, the helper functions can be supplied bytransient transfection of the cells with constructs that encode therequired helper functions, or the cells can be engineered to stablycontain genes encoding the helper functions, the expression of which canbe controlled at the transcriptional or posttranscriptional level. Inyet another system, the transgene flanked by ITRs and rep/cap genes areintroduced into insect cells by infection with baculovirus-basedvectors. For reviews on these production systems, see generally, e.g.,Grieger & Samulski, 2005; and Btining et at, 2008; Zhang et ai., 2009,“Adenovirus-adeno-associated virus hybrid for large-scale recombinantadeno-associated virus production,” Human Gene Therapy 20:922-929, thecontents of each of which is incorporated herein by reference in itsentirety. Methods of making and using these and other AAV productionsystems are also described in the following U.S. patents, the contentsof each of which is incorporated herein by reference in its entirety:U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213;6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898;7,229,823; and 7,439,065. See also the paragraphs below, which describemethods for scaling up AAV production using these systems and variantsthereof.

Due to size constraints of AAV for packaging (tolerating a transgene ofapproximately 4.5 kb), the transcription unites) (i.e., the transgeneunit, the ablator unit, and the dimerizable transcription factor unit)described may need to be engineered and packaged into two or morereplication-deficient AAV stocks. This may be preferable, because thereis evidence that exceeding the packaging capacity may lead to thegeneration of a greater number of “empty” AAV particles.

Alternatively, the available space for packaging may be conserved bycombining more than one transcription unit into a single construct, thusreducing the amount of required regulatory sequence space. For example,a single promoter may direct expression of a single RNA that encodes twoor three or more genes of interest, and translation of the downstreamgenes are driven by IRES sequences. In another example, a singlepromoter may direct expression of an RNA that contains, in a single openreading frame (ORF), two or three or more genes of interest separatedfrom one another by sequences encoding a self-cleavage peptide (e.g.,T2A) or a protease recognition site (e.g., furin). The ORF thus encodesa single polyprotein, which, either during (in the case of T2A) or aftertranslation, is cleaved into the individual proteins (such as, e.g.,transgene and dimerizable transcription factor). It should be noted,however, that although these IRES and polyprotein systems can be used tosave AAV packaging space, they can only be used for expression ofcomponents that can be driven by the same promoter.

In another alternative, the transgene capacity of AAV can be increasedby providing AAV ITRs of two genomes that can anneal to form head totail concatamers. Generally, upon entry of the AAV into the host cell,the single-stranded DNA containing the transgene is converted by hostcell DNA polymerase complexes into double-stranded DNA, after which theITRs aid in concatamer formation in the nucleus. As an alternative, theAAV may be engineered to be a self-complementary (sc) AAV, which enablesthe virus to bypass the step of second-strand synthesis upon entry intoa target cell, providing an scAAV virus with faster and, potentially,higher (e.g., up to 100-fold) transgene expression. For example, the AAVmay be engineered to have a genome comprising two connectedsingle-stranded DNAs that encode, respectively, a transgene unit and itscomplement, which can snap together following delivery into a targetcell, yielding a double-stranded DNA encoding the transgene unit ofinterest. Self-complementary AAV s are described in, e.g., U.S. Pat.Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporatedherein by reference in its entirety.

The transcription units(s) in the replication-deficient rAAVs may bepackaged with any AAV capsid protein (Cap) described herein, known inthe art, or to be discovered. Caps from serotypes AAV1, AAV6, AAV7,AAV8, AAV9 or rh10 are particularly preferred for generating rAAVs foruse in human subjects. In a preferred embodiment, an rAAV Cap is basedon serotype AAV8. In another embodiment, an rAAV Cap is based on Capsfrom two or three or more AAV serotypes. For example, in one embodiment,an rAAV Cap is based on AAV6 and AAV9.

Cap proteins have been reported to have effects on host tropism, cell,tissue, or organ specificity, receptor usage, infection efficiency, andimmunogenicity of AAV viruses. See, e.g., Grieger & Samulski, 2005;Buning et al., 2008; and the references cited below in this sub-section;all of which are incorporated herein by reference in their entirety.Accordingly, an AAV Cap for use in an rAAV may be selected based onconsideration of, for example, the subject to be treated (e.g., human ornon-human, the subject's immunological state, the subject's suitabilityfor long or short-term treatment, etc.) or a particular therapeuticapplication (e.g., treatment of a particular disease or disorder, ordelivery to particular cells, tissues, or organs).

In some embodiments, an rAAV Cap is selected for its ability toefficiently transduce a particular cell, tissue, or organ, for example,to which a particular therapy is targeted. In some embodiments, an rAAVCap is selected for its ability to cross a tight endothelial cellbarrier, for example, the blood-brain barrier, the blood-eye barrier,the blood-testes barrier, the blood-ovary barrier, the endothelial cellbarrier surrounding the heart, or the blood-placenta barrier.

Tissue specificity of adeno-associated viruses (AAV) serotypes isdetermined by the serotype of the capsid, and viral vector based ondifferent AAV capsids may generated taking into consideration theirability to infect different tissues. AAV2 presents a natural tropismtowards skeletal muscles, neurons of the central nervous system,vascular smooth muscle cells. AAV1 has been described as being moreefficient than AAV2 in transducing muscle, arthritic joints, pancreaticislets, heart, vascular endothelium, central nervous system (CNS) andliver cells, whereas AAV3 appears to be well suited for the transductionof cochlear inner hair cells, AAV4 for brain, AAV5 for CNS, lung, eye,arthritic joints and liver cells, AAV6 for muscle, heart and airwayepithelium, AAV7 for muscle, AAV8 for muscle, pancreas, heart and liver,and AAV9 for heart. See, e.g., Buning et at., 2008. Any serotype of AAVknown in the art,e.g., serotypes AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5,AAV6, AAV7 [see, WO 2003/042397], AAV8 [see, e.g., U.S. Pat. No.7,790,449; U.S. Pat. No. 7,282,199], AAV9 [see, WO 2005/033321], AAV10,AAV 11, AAV12, rh10, modified AAV [see, e.g., WO 2006/110689], or yet tobe discovered, or a recombinant AAV based thereon, may be used as asource for the rAAV capsid.

Various naturally occurring and recombinant AAVs, their encoding nucleicacids, AAV Cap and Rep proteins and their sequences, as well as methodsfor isolating or generating, propagating, and purifying such AAV s, andin particular, their capsids, suitable for use in producing rAAV s aredescribed in Gao et al., 2004, “Clades of adeno-associated viruses arewidely disseminated in human tissues,” J. Virol. 78:6381-6388; U.S. Pat.Nos. 7,319,002; 7,056,502; 7,282,199; 7,198,951; 7,235,393; 6,156,303;and 7,220,577; U.S. Patent Application Publication Nos. US 2003-0138772;US 2004-0052764; US 2007-0036760; US 2008-0075737; and US 2008-0075740;and International Patent Application Publication Nos. WO 20031014367; WO20011083692; WO 2003/042397 (AAV7 and various simian AAV); WO2003/052052; WO 2005/033321; WO 20061110689; WO 2008/027084; and WO2007/127264; each of which is incorporated herein by reference in itsentirety.

In some embodiments, an AAV Cap for use in the rAAV can be generated bymutagenesis (i. e., by insertions, deletions, or substitutions) of oneof the aforementioned AAV Caps or its encoding nucleic acid. In someembodiments, the AAV Cap is at least 70% identical, 75% identical, 80%identical, 85% identical, 90% identical, 95% identical, 98% identical,or 99% or more identical to one or more of the aforementioned AAV Caps.

In some embodiments, the AAV Cap is chimeric, comprising domains fromtwo or three or four or more of the aforementioned AAV Caps. In someembodiments, the AAV Cap is a mosaic of Vpl, Vp2, and Vp3 monomers fromtwo or three different AAVs or recombinant AAVs. In some embodiments, anrAAV composition comprises more than one of the aforementioned Caps.

In some embodiments, an AAV Cap for use in an rAAV composition isengineered to contain a heterologous sequence or other modification. Forexample, a peptide or protein sequence that confers selective targetingor immune evasion may be engineered into a Cap protein. Alternatively orin addition, the Cap may be chemically modified so that the surface ofthe rAAV is polyethylene glycolated (PEGylated), which may facilitateimmune evasion. The Cap protein may also be mutagenized, e.g., to removeits natural receptor binding, or to mask an immunogenic epitope.

Methods for Scalable Manufacture of AAV

Methods for the scalable (e.g., for production at commercial scale)manufacture of AAV, which may be adapted in order to generate rAAVcompositions that are suitably homogeneous and free of contaminants foruse in clinical applications, are also known in the art, and aresummarized briefly below.

Adeno-associated viruses can be manufactured at scale using a mammaliancell line-based approach, such as the approach using stable producercell lines described in Thome et al., 2009, “Manufacturing recombinantadeno-associated viral vectors from producer cell clones,” Human GeneTherapy 20:707-714, which is incorporated herein by reference in itsentirety. In the approach described by Thorpe and colleagues, producercell lines stably containing all the components needed to generate anrAAV—the transgene construct (transgene flanked by ITRs) and AAV rep andcap genes—are engineered, which are induced to make virus by infectionwith a helper virus, such as a live adenovirus type 5 (Ad5) (methods ofscalable production of which are also well known in the art). Producercell lines are stably transfected with construct(s) containing (i) apackaging cassette (rep and cap genes of the desired serotype andregulatory elements required for their expression), (ii) the transgeneflanked by ITRs, (iii) a selection marker for mammalian cells, and (iv)components necessary for plasmid propagation in bacteria. Stableproducer cell lines are obtained by transfecting the packagingconstruct(s), selecting drug-resistant cells, and replica-plating toensure production of the recombinant AAV in the presence of helpervirus, which are then screened for performance and quality. Onceappropriate clones are chosen, growth of the cell lines is scaled up,the cells are infected with the adenovirus helper, and resulting rAAVsare harvested from the cells.

In an alternative to the methods described in Thorpe et al., a packagingcell line is stably transfected with the AAV rep and cap genes, and thetransgene construct is introduced separately when production of the rAAVis desired. Although Thorpe and colleagues use HeLa cells for theproducer cell line, any cell line (e.g., Vero, A549, HEK 293) that issusceptible to infection with helper virus, able to maintain stablyintegrated copies of the rep gene and, preferably, able to grow well insuspension for expansion and production in a bioreactor may be used inaccordance with the methods described in Thorpe et al.

In the foregoing methods, rAAVs are produced using adenovirus as ahelper virus. In a modification of these methods, rAAV s can begenerated using producer cells stably transfected with one or moreconstructs containing adenovirus helper functions, avoiding therequirement to infect the cells with adenovirus. In a variation, one ormore of the adenovirus helper functions are contained within the sameconstruct as the rep and cap genes. In these methods, expression of theadenovirus helper functions may be placed under transcriptional orpost-transcriptional control to avoid adenovirus-associatedcytotoxicity.

In an alternative to producing stable cell lines, AAV s may also beproduced at scale using transient transfection methods, such asdescribed by Wright, 2009, “Transient transfection methods for clinicaladeno-associated viral vector production,” Human Gene Therapy20:698-706, which is incorporated herein by reference in its entirety.Wright's approach involves transfection of cells with constructs thatcontain (i) the transgene of interest flanked by ITRs; (ii) the AAV repand cap genes; and (iii) helper virus (e.g., adenovirus) genes requiredto support genome replication and packaging (or alternatively, a helpervirus, as described in Thorpe et al.), Alternatively, the adenovirushelper functions may be contained within the same construct as the repand cap genes. Thus, rAAV s are produced without having to ensure stabletransfection of the transgene and rep/cap constructs. This provides aflexible and quick method for generating AAV s, and is thus ideal forpre-clinical and early-phase clinical development. Recombinant AAVs canbe generated by transiently transfecting mammalian cell lines with theconstructs using transient transfection methods known in the art. Forexample, transfection methods most suited for large-scale productioninclude DNA co-precipitation with calcium phosphate, the use ofpoly-cations such as polyethylenimine (PE), and cationic lipids.

The effectiveness of adenovirus as a helper has also been exploited todevelop alternative methods for large-scale recombinant AAV production,for example using hybrid viruses based on adenovirus and AAV (an “Ad-AAVhybrid”). This production method has the advantage that it does notrequire transfection—all that is required for rAAV production isinfection of the rep/cap packaging cells by adenoviruses. In thisprocess, a stable rep/cap cell line is infected with a helper adenoviruspossessing functional E 1 genes and, subsequently, a recombinant Ad-AAVhybrid virus in which the AAV transgene plus ITRs sequence is insertedinto the adenovirus El region. Methods for generating Ad-AAV hybrids andtheir use in recombinant AAV production are described in Zhang et al.,2009, which is incorporated by reference herein in its entirety.

In another variation, rAAVs can be generated using hybrid viruses basedon AAV and herpes simplex virus type 1 (HSV) (an “HSV/AAV hybrid”), suchas described in Clement et al., 2009, “Large-scale adeno-associatedviral vector production using a herpesvirus-based system enablesmanufacturing for clinical studies,” Human Gene Therapy 20:796-806,which is incorporated herein by reference in its entirety. This methodexpands on the possibility of using HSV as a helper virus for AAVproduction (well known in the art, and also reviewed in Clement et al.).Briefly, HSV/AAV hybrids comprise an AAV transgene construct within anHSV backbone. These hybrids can be used to infect producer cells thatsupply the rep/cap and herpesvirus helper functions, or can be used inco-infections with recombinant HSV s that supply the helper functions,resulting in generation of rAAV s encapsidating the transgene ofinterest.

In another method, rAAV compositions may produced at scale usingrecombinant baculovirus-mediated expression of AAV components in insectcells, for example, as described in Virag et al., 2009, “Producingrecombinant adeno-associated virus in foster cells: Overcomingproduction limitations using a baculovirus-insect cell expressionstrategy,” Human Gene Therapy 20:807-817, which is incorporated hereinby reference in its entirety. In this system, the well-known baculovirusexpression vector (BEV) system is adapted to produce recombinant AAVs.For example, the system described by Virag et al. comprises theinfection of Sf9 insect cells with two (or three) different BEVs thatprovide (i) AAV rep and cap (either in one or two BEVs) and (ii) thetransgene construct. Alternatively, the Sf9 cells can be stablyengineered to express rep and cap, allowing production of recombinantAAV s following infection with only a single BEV containing thetransgene construct. In order to ensure stoichiometric production of theRep and Cap proteins, the latter of which is required for efficientpackaging, the BEV s can be engineered to include features that enablepre- and post-transcriptional regulation of gene expression. The Sf9cells then package the transgene construct into AAV capsids, and theresulting rAAV can be harvested from the culture supernatant or bylysing the cells.

Each of the foregoing methods permit the scalable production of rAAVcompositions. The manufacturing process for an rAAV composition suitablefor commercial use (including use in the clinic) must also comprisesteps for removal of contaminating cells; removing and inactivatinghelper virus (and any other contaminating virus, such as endogenousretrovirus-like particles); removing and inactivating any rcAAV;minimizing production of, quantitating, and removing empty(transgene-less) AAV particles (e.g., by centrifugation); purifying therAAV (e.g., by filtration or chromatography based on size and/oraffinity); and testing the rAAV composition for purity and safety. Thesemethods are also provided in the references cited in the foregoingparagraphs and are incorporated herein for this purpose.

One disadvantage of the foregoing methods of scalable rAAV production isthat much of the rAAV is obtained by lysing the producer cells, whichrequires significant effort to not only obtain the virus but also toisolate it from cellular contaminants To minimize these requirements,scalable methods of rAAV production that do not entail cell lysis may beused, such as provided in International Patent Application PublicationNo. WO 2007/127264, the contents of which is incorporated by referenceherein in its entirety. In the example of Section 6 infra, a newscalable method obtaining rAAV from cell culture supernatants isprovided, which may also be adapted for the preparation of rAAVcomposition for use in accordance with the methods described herein.

In still another embodiment, the invention provides human or non-humancells which contain one or more of the DNA constructs and/or viruscompositions of the invention. Such cells may be genetically engineeredand may include, e.g., plant, bacterial, non-human mammalian ormammalian cells. Selection of the cell types is not a limitation of theinvention.

5.2. Compositions

The present invention provides replication-defective virus compositionssuitable for use in therapy (in vivo or ex vivo)in which the genome ofthe virus (or the collective genomes of two or morereplication-defective virus stocks used in combination) comprise thetherapeutic transgene unit and the ablator unit defined in Section 3.1,and described supra; and may further comprise dimerizable fusion proteinor TF domain units(s) (referred to for purposes of convenience asdimerizable unit(s)). Any virus suitable for gene therapy may be used inthe compositions of the invention, including but not limited toadeno-associated virus (“AAV”), adenovirus, herpes simplex virus,lentivirus, or a retrovirus. In a preferred embodiment, the compositionsare replication-defective AAV s, which are described in more detail inSection 5.2.1 herein.

The compositions of the invention comprise a replication-defectivevirus(es) suitable for therapy (in vivo or ex vivo) in which the genomeof the virus(es) comprises a transgene unit, an ablation unit, and/or adimerizable unit. In one embodiment, a composition of the inventioncomprises a virus suitable for gene therapy in which the genome of thevirus comprises a transgene unit. In another embodiment, a compositionof the invention comprises a virus suitable for gene therapy in whichthe genome of the virus comprises an ablation unit. In anotherembodiment, a composition of the invention comprises a virus suitablefor gene therapy in which the genome of the virus comprises adimerizable unit. In another embodiment, a composition of the inventioncomprises a virus suitable for gene therapy in which the genome of thevirus comprises a transgene unit and an ablation unit. In anotherembodiment, a composition of the invention comprises a virus suitablefor gene therapy in which the genome of the virus comprises a transgeneunit and a dimerizable unit. In another embodiment, a composition of theinvention comprises a virus suitable for gene therapy in which thegenome of the virus comprises an ablation unit and a dimerizable unit.In another embodiment, a composition of the invention comprises virusessuitable for gene therapy in which the genome of the virus comprises atransgene unit, an ablation unit and a dimerizable unit.

The invention also provides compositions comprising recombinant DNAconstructs that comprise one or more transcriptional units describedherein. Compositions comprising recombinant DNA constructs are describedin more detail in Section 5.2.2.

5.2.1. Replication-Defective Virus Compositions for Gene Therapy

The invention provides compositions comprising a replication-defectivevirus stock(s) and formulations of the replication-defective virus(es)in a physiologically acceptable carrier. These formulations can be usedfor gene transfer and/or gene therapy. The viral genome of thecompositions comprises: (a) a first transcription unit that encodes atherapeutic product in operative association with a promoter thatcontrols transcription, said unit containing at least one ablationrecognition site (transgene unit); and (b) a second transcription unitthat encodes an ablator specific for the ablation recognition site, or afragment thereof, in operative association with a promoter. In oneembodiment, the viral genome of the replication-defective virus. Theablator is as defined elsewhere in this specification.

AAV Stocks

In a preferred embodiment, the replication-defective virus of acomposition of the invention is an AAV, preferably AAV1, AAV6, AAV6.2,AAV7, AAV8, AAV9 or rh10. In one embodiment, the AAV of the compositionis AAV8. Due to the packaging constraints of AAV (approximately 4.5 kb)in most cases, for ease of manufacture, the transgene unit, the ablationunit, and the dimerizable unit will be divided between two or more viralvectors and packaged in a separate AAV stock. In one embodiment, thereplication-defective virus composition comprises the firsttranscription unit (a transgene unit) packaged in one AAV stock, and thesecond (an ablator unit), third and fourth transcription units(dimerizable TF domain unit) packaged in a second AAV stock. In anotherembodiment, the replication-defective virus composition comprises thesecond transcription unit (an ablator unit) packaged in one AAV stock,and the first (a transgene unit), third and fourth transcription units(dimerizable TF domain unit) packaged in a second AAV stock. In anotherembodiment, all four units can be packaged in one AAV stock, but thisimposes limits on the size of the DNAs that can be packaged. Forexample, when using Cre as the ablator and FRB/FKB as the dimerizable TFdomains (as shown in the examples, infra), in order to package all fourunits into one AAV stock, the size of the DNA encoding the therapeutictransgene should be less than about 900 base pairs in length; this wouldaccommodate DNAs encoding cytokines, RNAi therapeutics, and the like.

Due to size constraints of the AAV genome for packaging, thetranscription units can be engineered and packaged in two or more AAVstocks. Whether packaged in one viral stock which is used as a viruscomposition according to the invention, or in two or more viral stockswhich form a virus composition of the invention, the viral genome usedfor treatment must collectively contain the first and secondtranscription units encoding the therapeutic transgene and the ablator;and may further comprise additional transcription units (e.g., the thirdand fourth transcription units encoding the dimerizable TF domains). Forexample, the first transcription unit can be packaged in one viralstock, and second, third and fourth transcription units packaged in asecond viral stock. Alternatively, the second transcription unit can bepackaged in one viral stock, and the first, third and fourthtranscription units packaged in a second viral stock. While useful forAAV due to size contains in packaging the AAV genome, other viruses maybe used to prepare a virus composition according to the invention. Inanother embodiment, the viral compositions of the invention, where theycontain multiple viruses, may contain different replication-defectiveviruses (e.g., AAV and adenovirus).

In one embodiment, a virus composition according to the inventioncontains two or more different AAV (or another viral) stock, in suchcombinations as are described above. For example, a virus compositionmay contain a first viral stock comprising the therapeutic gene withablator recognition sites and a first ablator and a second viral stockcontaining an additional ablator(s). Another viral composition maycontain a first virus stock comprising a therapeutic gene and a fragmentof an ablator and a second virus stock comprising another fragment of anablator. Various other combinations of two or more viral stocks in avirus composition of the invention will be apparent from the descriptionof the components of the present system.

Viral Formulations

Compositions of the invention may be formulated for delivery to animalsfor veterinary purposes (e.g., livestock (cattle, pigs, etc), and othernon-human mammalian subjects, as well as to human subjects. Thereplication-defective viruses can be formulated with a physiologicallyacceptable carrier for use in gene transfer and gene therapyapplications. Because the viruses are replication-defective, the dosageof the formulation cannot be measured or calculated as a PFU (plaqueforming unit). Instead, quantification of the genome copies (“GC”) maybe used as the measure of the dose contained in the formulation.

Any method known in the art can be used to determine the genome copy(GC) number of the replication-defective virus compositions of theinvention. One method for performing AAV GC number titration is asfollows: Purified AAV vector samples are first treated with DNase toeliminate un-encapsidated AAV genome DNA or contaminating plasmid DNAfrom the production process. The DNase resistant particles are thensubjected to heat treatment to release the genome from the capsid. Thereleased genomes are then quantitated by real-time PCR usingprimer/probe sets targeting specific region of the viral genome (usuallypoly A signal).

Also, the replication-defective virus compositions can be formulated indosage units to contain an amount of replication-defective virus that isin the range of about 1.0×10⁹ GC to about 1.0×10¹⁵ GC (to treat anaverage subject of 70 kg in body weight), and preferably 1.0×10¹² GC to1.0×10¹⁴ GC for a human patient. Preferably, the dose ofreplication-defective virus in the formulation is 1.0×10⁹ GC, 5.0×10⁹GC, 1.0×10¹⁰ GC, 5.0×10¹⁰ GC, 1.0×10¹¹ GC, 5.0×10¹¹ GC, 1.0×10¹² GC,5.0×10¹² GC, or 1.0×10¹³ GC, 5.0×10¹³ GC, 1.0×10¹⁴ GC, 5.0×10¹⁴ GC, or1.0×10¹⁵ GC.

The replication-defective viruses can be formulated in a conventionalmanner using one or more physiologically acceptable carriers orexcipients. The replication-defective viruses may be formulated forparenteral administration by injection, e.g., by bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The replication-defective virus compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Liquid preparations of thereplication-defective virus formulations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts. Alternatively, the compositions may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

Also encompassed is the use of adjuvants in combination with or inadmixture with the replication-defective viruses of the invention.Adjuvants contemplated include but are not limited to mineral saltadjuvants or mineral salt gel adjuvants, particulate adjuvants,microparticulate adjuvants, mucosal adjuvants, and immunostimulatoryadjuvants. Adjuvants can be administered to a subject as a mixture withreplication-defective viruses of the invention, or used in combinationwith the replication-defective viruses of the invention.

5.2.2. Recombinant DNA Construct Compositions For Production ofReplication-Defective Viral Vectors Useful for Therapeutic Purposes

The invention provides recombinant DNA construct compositions comprisinga transgene unit, an ablation unit, and/or one or two dimerizable domainunits flanked by viral signals that define the region to be amplifiedand packaged into replication-defective viral particles. These DNAconstructs can be used to generate the replication-defective viruscompositions and stocks.

In one embodiment, the recombinant DNA construct comprises a transgeneunit flanked by packaging signals of a viral genome. In anotherembodiment, a composition of the invention comprises a recombinant DNAconstruct comprising an ablation unit flanked by packaging signals of aviral genome. In another embodiment, the recombinant DNA constructcomprises a dimerizable unit flanked by packaging signals of a viralgenome. In another embodiment, the recombinant DNA construct comprises atransgene unit and an ablation unit flanked by packaging signals of aviral genome. In another embodiment, the recombinant DNA constructcomprises a transgene unit and a dimerizable unit flanked by packagingsignals of a viral genome. In another embodiment, the recombinant DNAconstruct comprises an ablation unit and a dimerizable unit flanked bypackaging signals of a viral genome. In another embodiment, therecombinant DNA construct comprises a transgene unit, an ablation unitand a dimerizable unit flanked by packaging signals of a viral genome.

The first transcription unit encodes a therapeutic product in operativeassociation with a promoter that controls transcription, said unitcontaining at least one ablation recognition site (transgene unit); and(b) the second transcription unit that encodes an ablator specific forthe ablation recognition site, or a fragment thereof fused to a bindingdomain, in operative association with a promoter that inducestranscription in response to a pharmacological agent (ablation unit). Inanother embodiment, the recombinant DNA construct comprises adimerizable TF domain unit flanked by packaging signals of a viralgenome.

In a preferred embodiment, the recombinant DNA construct compositionfurther comprises a dimerizable unit nested within the viral packagingsignals. In one embodiment, each unit encodes a dimerizable domain of atranscription factor that regulates the inducible promoter of the secondtranscription unit, in which (c) a third transcription unit encodes theDNA binding domain of the transcription factor fused to a binding domainfor the pharmacological agent in operative association with aconstitutive promoter; and (d) a fourth transcription unit encodes theactivation domain of the transcription factor fused to a binding domainfor the pharmacological agent in operative association with aconstitutive promoter. In another embodiment, at least one of (c) or (d)is expressed under an inducible promoter. In a specific embodiment, thepharmacological agent that induces transcription of the promoter that isin operative association with the second unit of the recombinant DNAconstruct composition is a dimerizer that dimerizes the domains of thetranscription factor as measured in vitro. In yet another specificembodiment, the pharmacological agent that induces transcription of thepromoter that is in operative association with the second unit of therecombinant DNA construct composition is rapamycin. In still a furtherembodiment, the recombinant DNA construct comprises a dimerizable fusionprotein unit. For example, the dimerizable fusion protein unit may beencode (a) a binding domain of an enzyme fused to a binding domain and(b) a catalytic domain of the enzyme fused to a binding domain, wherethe binding domains are either DNA binding domains or the bindingdomains for a dimerizer.

In order to conserve space within the viral genome(s), bicistronictranscription units can be engineered. For example, the third and fourthtranscription units can be engineered as a bicistronic unit containingan IRES (internal ribosome entry site), which allows coexpression ofheterologous gene products by a message from a single promoter.Altenatively, a single promoter may direct expression of an RNA thatcontains, in a single open reading frame (ORF), two or threeheterologous genes (e.g., the third and fourth transcription units)separated from one another by sequences encoding a self-cleavage peptide(e.g., T2A) or a protease recognition site (e.g., furin). The ORF thusencodes a single polyprotein, which, either during (in the case of T2A)or after translation, is cleaved into the individual proteins. It shouldbe noted, however, that although these IRES and polyprotein systems canbe used to save AAV packaging space, they can only be used forexpression of components that can be driven by the same promoter.

In a specific embodiment, a recombinant DNA construct composition thatcomprises a dimerizable unit comprises an IRES. In another specificembodiment, a recombinant DNA construct composition that comprises athird and fourth transcription unit (a dimerizable TF domain unit)comprises and IRES In another specific embodiment, a recombinant DNAconstruct composition that comprises a transgene unit comprises an IRES.In another specific embodiment, a recombinant DNA construct compositionthat comprises an ablation unit comprises an IRES. In another specificembodiment, a recombinant DNA construct composition that comprises adimerizable unit comprises an IRES.

In a specific embodiment, a recombinant DNA construct composition thatcomprises a third and a fourth transcription unit (a dimerizable TFdomain unit) comprises T2A sequence. In another specific embodiment, arecombinant DNA construct composition that comprises a transgene unitcomprises T2A sequence. In another specific embodiment, a recombinantDNA construct composition that comprises an ablation unit comprises T2Asequence. In another specific embodiment, a recombinant DNA constructcomposition that comprises a dimerizable TF domain unit comprises T2Asequence.

In an embodiment, the ablator that is encoded by the secondtranscription unit of the recombinant DNA construct composition is anendonuclease, a recombinase, a meganuclease, or an artificial zincfinger endonuclease that binds to the ablation recognition site in thefirst transcription unit and excises or ablates DNA. In a specificembodiment, the ablator is ere and the ablation recognition site isLoxP, or the ablator is FLP and the ablation recognition site is FRT. Inanother embodiment, the ablator that is encoded by the second.transcription unit of the recombinant DNA construct composition is aninterfering RNA, a ribozyme, or an antisense that ablates the RNAtranscript of the first transcription unit, or suppresses translation ofthe RNA transcript of the first transcription unit. In a specificembodiment, transcription of the ablator is controlled by a tet-on/offsystem, a tetR-KRAB system, a mifepristone (RU486) regulatable system, atamoxifen-dep regulatable system, or an ecdysone-dep regulatable system.

The recombinant DNA construct composition contains packaging signalsflanking the transcription units desired to be amplified and packaged inreplication-defective virus vectors. In a specific embodiment, thepackaging signals are AAV ITRs. Where a pseudotyped AAV is to beproduced, the ITRs are selected from a source which differs from the AAVsource of the capsid. For example, AAV2 ITRs may be selected for usewith an AAV1, AAV8, or AAV9 capsid, and so on. In another specificembodiment, the AAV ITRs may be from the same source as the capsid,e.g., AAV1, AAV6, AAV7, AAV8, AAV9, rh10 ITRs, etc. In another specificembodiment, a recombinant DNA construct composition comprises a firsttranscription unit (transgene unit) flanked by AAV ITRs, and the second(ablation unit), and optional third and fourth transcription units (adimerizable TF domain unit), and/or a dimerizable fusion proteinunit(s), flanked by AAV ITRs. In yet another specific embodiment, arecombinant DNA construct composition comprises a second transcriptionunit (ablation unit) flanked by AAV ITRs, and the first (transgeneunit), third and fourth transcription units (a dimerizable TF domainunit) are flanked by AAV ITRs. In a preferred embodiment, thetranscription units of a PIT A system are contained in two or morerecombinant DNA compositions.

In a specific embodiment, recombinant DNA construct contains a transgeneunit that encodes anyone or more of the following therapeutic products:an antibody or antibody fragment that neutralizes HIV infectivity,soluble vascular endothelial growth factor receptor-1 (sFlt-I), FactorVIII, Factor IX, insulin like growth factor (IGF), hepatocyte growthfactor (HGF), heme oxygenase-1 (HO-1), or nerve growth factor (NGF). Ina specific embodiment, recombinant DNA construct contains a transgeneunit that comprises anyone of the following promoters that controlstranscription of the therapeutic gene: a constitutive promoter, atissue-specific promoter, a cell-specific promoter, an induciblepromoter, or a promoter responsive to physiologic cues.

The DNA constructs can be used in any of the methods described inSection 5.1.5 to generate replication-defective virus stocks.

5.2.3. Pharmaceutical Compositions and Formulations of Dimerizers

The present invention provides pharmaceutical compositions comprisingthe dimerizers of the invention, described in Section 5.1.4. In apreferred embodiment, the pharmaceutical compositions comprise apharmaceutically acceptable carrier or excipient. Optionally, thesepharmaceutical compositions are adapted for veterinary purposes, e.g.,for delivery to a non-human mammal (e.g., livestock), such as aredescribed herein.

The pharmaceutical compositions of the invention can be administered toa subject at therapeutically effective doses to ablate or excise thetransgene of a transgene unit of the invention or to ablate thetranscript of the transgene, or inhibit its translation. Atherapeutically effective dose refers to an amount of the pharmaceuticalcomposition sufficient to result in amelioration of symptoms caused byexpression of the transgene, e.g., toxicity, or to result in at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% inhibition of expressionof the transgene.

In an embodiment, an amount of pharmaceutical composition comprising adimerizer of the invention is administered that is in the range of about0.1-5 micrograms (μg)/kilogram (kg). To this end, a pharmaceuticalcomposition comprising a dimerizer of the invention is formulated indoses in the range of about 7 mg to about 350 mg to treat to treat anaverage subject of 70 kg in body weight. The amount of pharmaceuticalcomposition comprising a dimerizer of the invention administered is:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5 or 5.0 mg/kg. The dose of a dimerizer in a formulation is7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 90,95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750mg (to treat to treat an average subject of 70 kg in body weight). Thesedoses are preferably administered orally. These doses can be given onceor repeatedly, such as daily, every other day, weekly, biweekly, ormonthly. Preferably, the pharmaceutical compositions are given onceweekly for a period of about 4-6 weeks. In some embodiments, apharmaceutical composition comprising a dimerizer is administered to asubject in one dose, or in two doses, or in three doses, or in fourdoses, or in five doses, or in six doses or more. The interval betweendosages may be determined based the practitioner's determination thatthere is a need for inhibition of expression of the transgene, forexample, in order to ameliorate symptoms caused by expression of thetransgene, e.g., toxicity. For example, in some embodiments when theneed for transgene ablation is acute, daily dosages of a pharmaceuticalcomposition comprising a dimerizer may be administered. In otherembodiments, e.g., when the need for transgene ablation is less acute,or is not acute, weekly dosages of a pharmaceutical compositioncomprising a dimerizer may be administered.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the dimerizersand their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) oral, buccal, parenteral, rectal, ortransdermal administration. Noninvasive methods of administration arealso contemplated.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the dimerizers.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the dimerizers for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the dimerizersand a suitable powder base such as lactose or starch.

The dimerizers may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The dimerizers may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the dimerizers mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thedimerizers may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Also encompassed is the use of adjuvants in combination with or inadmixture with the dimerizers of the invention. Adjuvants contemplatedinclude but are not limited to mineral salt adjuvants or mineral saltgel adjuvants, particulate adjuvants, microparticulate adjuvants,mucosal adjuvants, and immunostimulatory adjuvants. Adjuvants can beadministered to a subject as a mixture with dimerizers of the invention,or used in combination with the dimerizers of the invention.

5.3. Treatment of Diseases and Disorders

The invention provides methods for treating any disease or disorder thatis amenable to gene therapy. In one embodiment, “treatment” or“treating” refers to an amelioration of a disease or disorder, or atleast one discernible symptom thereof. In another embodiment,“treatment” or “treating” refers to an amelioration of at least onemeasurable physical parameter associated with a disease or disorder, notnecessarily discernible by the subject. In yet another embodiment,“treatment” or “treating” refers to inhibiting the progression of adisease or disorder, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. Other conditions, including cancer, immunedisorders, and veterinary conditions, may also be treated.

5.3.1. Target Diseases

Types of diseases and disorders that can be treated by methods of thepresent invention include, but are not limited to age-related maculardegeneration; diabetic retinopathy; infectious diseases e.g., HIVpandemic flu, category 1 and 2 agents of biowarfare, or any new emergingviral infection; autoimmune diseases; cancer; multiple myeloma;diabetes; systemic lupus erythematosus (SLE); hepatitis C; multiplesclerosis; Alzheimer's disease; parkinson's disease; amyotrophic lateralsclerosis (ALS), huntington's disease; epilepsy; chronic obstructivepulmonary disease (COPD); joint inflammation, arthritis; myocardialinfarction (MI); congestive heart failure (CHF); hemophilia A; orhemophilia B.

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to, viruses, bacteria, fungi, protozoa, helminths, andparasites. The invention is not limited to treating or preventinginfectious diseases caused by intracellular pathogens. Many medicallyrelevant microorganisms have been described extensively in theliterature, e.g., see C. G. A Thomas, Medical Microbiology, BailliereTindall, Great Britain 1983, the entire contents of which are herebyincorporated herein by reference.

Bacterial infections or diseases that can be treated or prevented by themethods of the present invention are caused by bacteria including, butnot limited to, bacteria that have an intracellular stage in its lifecycle, such as mycobacteria (e.g., Mycobacteria tuberculosis, M bovis, Mavium, M leprae, or M africanum), rickettsia, mycoplasma, chlamydia, andlegionella. Other examples of bacterial infections contemplated includebut are not limited to infections caused by Gram positive bacillus(e.g., Listeria, Bacillus such as Bacillus anthracia, Erysipelothrixspecies), Gram negative bacillus (e.g., Bartonella, Brucella,Campylobacter, Enterobacter, Escherichia, Francisella, Hemophilus,Klebsiella, Morganella, Proteus, Providencia, Pseudomonas, Salmonella,Serratia, Shigella, Vibrio, and Yersinia species), spirochete bacteria(e.g., Borrelia species including Borrelia burgdorferi that causes Lymedisease), anaerobic bacteria (e.g., Actinomyces and Clostridiumspecies), Gram positive and negative coccal bacteria, Enterococcusspecies, Streptococcus species, Pneumococcus species, Staphylococcusspecies, Neisseria species. Specific examples of infectious bacteriainclude but are not limited to: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria tuberculosis, Mavium, M intracellulare, M kansaii, M gordonae, Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis,Streptococcus bovis, Streptococcus pneumoniae, Haemophilus injluenzae,Bacillus antracis, corynebacterium diphtheriae, Erysipelothrixrhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacteraerogenes, Klebsiella pneumoniae, Pasturella multocida, Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Infectious virus of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses. Examples of virus that havebeen found in humans include but are not limited to: Retroviridae (e.g.human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LA V or HTLV-III/LA V, or HIV-III; and other isolates, such asHIV-LP; Picomaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses);Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus,respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);Bungaviridae, (e.g. Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g. reoviruses, orbiviurses and rotaviruses); Bimaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae(variola viruses, vaccinia viruses, pox viruses); and lridoviridae (e.g.African swine fever virus); and unclassified viruses (e.g. theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and relatedviruses, and astroviruses).

Parasitic diseases that can be treated or prevented by the methods ofthe present invention including, but not limited to, amebiasis, malaria,leishmania, coccidia, giardiasis, cryptosporidiosis, toxoplasmosis, andtrypanosomiasis. Also encompassed are infections by various worms, suchas but not limited to ascariasis, ancylostomiasis, trichuriasis,strongyloidiasis, toxoccariasis, trichinosis, onchocerciasis, filaria,and dirofilariasis. Also encompassed are infections by various flukes,such as but not limited to schistosomiasis, paragonimiasis, andclonorchiasis. Parasites that cause these diseases can be classifiedbased on whether they are intracellular or extracellular. An“intracellular parasite” as used herein is a parasite whose entire lifecycle is intracellular. Examples of human intracellular parasitesinclude Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Toxoplasmagondii, Babesia spp., and Trichinella spiralis. An “extracellularparasite” as used herein is a parasite whose entire life cycle isextracellular. Extracellular parasites capable of infecting humansinclude Entamoeba histolytica, Giardia lamblia, Enterocytozoon bieneusi,Naegleria and Acanthamoeba as well as most helminths Yet another classof parasites is defined as being mainly extracellular but with anobligate intracellular existence at a critical stage in their lifecycles. Such parasites are referred to herein as “obligate intracellularparasites”. These parasites may exist most of their lives or only asmall portion of their lives in an extracellular environment, but theyall have at least one obligate intracellular stage in their life cycles.This latter category of parasites includes Trypanosoma rhodesiense andTrypanosoma gambiense, Isospora spp., Cryptosporidium spp, Eimeria spp.,Neospora spp., Sarcocystis spp., and Schistosoma spp.

Types of cancers that can be treated or prevented by the methods of thepresent invention include, but are not limited to human sarcomas andcarcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease.

5.3.2. Dosage and Mode of Administration of Viral Vectors

The replication-defective virus compositions of the invention can beadministered to a human subject by any method or regimen known in theart. For example, the replication-defective virus compositions of theinvention can be administered to a human subject by any method describedin the following patents and patent applications that relate to methodsof using AAV vectors in various therapeutic applications: U.S. Pat. Nos.7,282,199; 7,198,951; U.S. Patent Application Publication Nos. US2008-0075737; US 2008-0075740; International Patent ApplicationPublication Nos. WO 2003/024502; WO 2004/108922; WO 20051033321, each ofwhich is incorporated by reference in its entirety.

In an embodiment, the replication-defective virus compositions of theinvention are delivered systemically via the liver by injection of amesenteric tributary of portal vein. In another embodiment, thereplication-defective virus compositions of the invention are deliveredsystemically via muscle by intramuscular injection in to e.g., thequadriceps or bicep muscles. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the basal forebrain region of the brain containing the nucleusbasalis of Meynert (NBM) by bilateral, stereotactic injection. Inanother embodiment, the replication-defective virus compositions of theinvention are delivered to the eNS by bilateral intraputaminal and/orintranigral injection. In another embodiment, the replication-defectivevirus compositions of the invention are delivered to the joints byintraarticular injection. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the heart by intracoronary infusion. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the retina by injection into the subretinal space.

In another embodiment, an amount of replication-defective viruscomposition is administered at an effective dose that is in the range ofabout 1.0×10⁸ genome copies (GC)/kilogram (kg) to about 1.0×10¹⁴ GC/kg,and preferably 1.0×10¹¹ GC/kg to 1.0×10¹³ GC/kg to a human patient.Preferably, the amount of replication-defective virus compositionadministered is 1.0×10⁸ GC/kg, 5.0×10⁸ GC/kg, 1.0×10⁹ GC/kg, 5.0×10⁹GC/kg, 1.0×10¹⁰ GC/kg, 5.0×10¹⁰ GC/kg, 1.0×10¹¹ GC/kg, 5.0×10¹¹ GC/kg,or 1.0×10¹² GC/kg, 5.0×10¹² GC/kg, 1.0×10¹³ GC/kg, 5.0×10¹³ GC/kg,1.0×10¹⁴ GC/kg

These doses can be given once or repeatedly, such as daily, every otherday, weekly, biweekly, or monthly, or until adequate transgeneexpression is detected in the patient. In an embodiment,replication-defective virus compositions are given once weekly for aperiod of about 4-6 weeks, and the mode or site of administration ispreferably varied with each administration. Repeated injection is mostlikely required for complete ablation of transgene expression. The samesite may be repeated after a gap of one or more injections. Also, splitinjections may be given. Thus, for example, half the dose may be givenin one site and the other half at another site on the same day.

When packaged in two or more viral stocks, the replication-defectivevirus compositions can be administered simultaneously or sequentially.When two or more viral stocks are delivered sequentially, the laterdelivered viral stocks can be delivered one, two, three, or four daysafter the administration of the first viral stock. Preferably, when twoviral stocks are delivered sequentially, the second delivered viralstock is delivered one or two days after delivery of the first viralstock.

Any method known in the art can be used to determine the genome copy(GC) number of the replication-defective virus compositions of theinvention. One method for performing AAV GC number titration is asfollows: Purified AAV vector samples are first treated with DNase toeliminate un-encapsidated AAV genome DNA or contaminating plasmid DNAfrom the production process. The DNase resistant particles are thensubjected to heat treatment to release the genome from the capsid. Thereleased genomes are then quantitated by real-time PCR usingprimer/probe sets targeting specific region of the viral genome (usuallypoly A signal).

In one embodiment, the replication-defective virus compositions of theinvention are delivered systemically via the liver by injection of amesenteric tributary of portal vein at a dose of about 3.0×10¹² GC/kg.In another embodiment, the replication-defective virus compositions ofthe invention are delivered systemically via muscle by up to twentyintramuscular injections in to either the quadriceps or bicep muscles ata dose of about 5.0×10¹² GC/kg. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the basal forebrain region of the brain containing the nucleusbasalis of Meynert (NBM) by bilateral, stereotactic injection at a doseof about 5.0×10¹¹ GC/kg. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the CNS by bilateral intraputaminal and/or intranigral injection at adose in the range of about 1.0×10¹¹ GC/kg to about 5.0×10¹¹ GC/kg. Inanother embodiment, the replication-defective virus compositions of theinvention are delivered to the joints by intra-articular injection at adose of about 1.0×1011 GC/mL of joint volume for the treatment ofinflammatory arthritis. In another embodiment, the replication-defectivevirus compositions of the invention are delivered to the heart byintracoronary infusion injection at a dose in the range of about1.4×10¹¹ GC/kg to about 3.0×10¹² GC/kg. In another embodiment, thereplication-defective virus compositions of the invention are deliveredto the retina by injection into the subretinal space at a dose of about1.5×10¹⁰ GC/kg.

Table 4 shows examples of transgenes that can be delivered via aparticular tissue/organ by the PITA system of the invention to treat aparticular disease.

TABLE 4 Treatment of Diseases Disease Examples of transgenes TargetTissue Age relation macular s-FIt-1, an anti-VEGF Retina degenerationantibody such as bevacizumab (Avastin), ranibizumab (Lucentis), or adomain antibody (dAB) HIV a neutralizing antibody Muscle and/or liveragainst HIV Cancer Antiangiogenic agents (s- Muscle and/or liver Fit-I,an anti-VEGF antibody such as bevacizumab (Avastin), ranibizumab(Lucentis), or a domain antibody (dAB); cytokines that enhance tumorimmune responses, anti-EGFR, IFN Autoimmune diseases, e.g., Antibodiesthat interfere Muscle and/or liver arthritis, systemic lupus withresponses e.g., β-IFN; T cell activation; adhesion molecule a4-erythematosus, psoriasis, integrin antibody cytokines that bias immunemultiple sclerosis (MS) Multiple myeloma anti-CD20 antibody Muscleand/or liver Diabetes GLP-1, IL-6 Muscle and/or liver Hepatitis C β-IFN,shRNA targeting Muscle and/or liver IRES Alzheimer's disease NGF Centralnervous system (CNS) Amyotrophic lateral sclerosis IGF-1 CNS (ALS)Huntington's disease NGF, BDNF AND CNTF, CNS shRNA targeting mutantHuntington Epilepsy galanin, neuropeptide Y CNS (NPY), glial cell linederived neurotrophic factor (GDNF) COPD chemokines from IL 8 Lungfamily, TNF antagonist Inflammatory arthritis TNF antagonist, IL-1,Joint anti-CD 20, IL-6, IL-1r antagonist Myocardial infarction Hemeoxygenase-1 Heart Congestive heart failure insulin like growth factorHeart (IGF), hepatocyte growth factor (HGF) Parkinson's Disease GDNF,aromatic L-amino CNS acid decarboxylase (ADCC), NGF

In one embodiment a method for treating age-related macular degenerationin a human subject comprises administering an effective amount of areplication-defective virus composition, in which the therapeuticproduct is a VEGF antagonist.

In another embodiment, a method for treating hemophilia A in a humansubject, comprises administering an effective amount of areplication-defective virus composition, in which the therapeuticproduct is Factor VIII or its variants, such as the light chain andheavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No.6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for2351 amino acids and the protein has six domains, designated from theamino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al,Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Tooleet al, Nature, 342:337 (1984)]. Human Factor VIII is processed withinthe cell to yield a heterodimer primarily comprising a heavy chaincontaining the A1, A2 and B domains and a light chain containing the A3,C1 and C2 domains. Both the single chain polypeptide and the heterodimercirculate in the plasma as inactive precursors, until activated bythrombin cleavage between the A2 and B domains, which releases the Bdomain and results in a heavy chain consisting of the A1 and A2 domains.The B domain is deleted in the activated procoagulant form of theprotein. Additionally, in the native protein, two polypeptide chains(“a” and “b”), flanking the B domain, are bound to a divalent calciumcation. In some embodiments, the minigene comprises first 57 base pairsof the Factor VIII heavy chain which encodes the 10 amino acid signalsequence, as well as the human growth hormone (hGH) polyadenylationsequence. In alternative embodiments, the minigene further comprises theA 1 and A2 domains, as well as 5 amino acids from the N-terminus of theB domain, and/or 85 amino acids of the C-terminus of the B domain, aswell as the A3, C1 and C2 domains. In yet other embodiments, the nucleicacids encoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560]. Examples of naturallyoccurring and recombinant forms of Factor VII can be found in the patentand scientific literature including, U.S. Pat. No. 5,563,045, U.S. Pat.No. 5,451,521, U.S. Pat. No. 5,422,260, U.S. Pat. No. 5,004,803, U.S.Pat. No. 4,757,006, U.S. Pat. No. 5,661,008, U.S. Pat. No. 5,789,203,U.S. Pat. No. 5,681,746, U.S. Pat. No. 5,595,886, U.S. Pat. No.5,045,455, U.S. Pat. No. 5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat.No. 5,693,499, U.S. Pat. No. 5,587,310, U.S. Pat. No. 5,171,844, U.S.Pat. No. 5,149,637, U.S. Pat. No. 5,112,950, U.S. Pat. No. 4,886,876;International Patent Publication Nos. WO 94/11503, WO 87/07144, WO92/16557, WO 91/09122, WO 97/03195, WO 96/21035, and WO 91/07490;European Patent Application Nos. EP 0 672 138, EP 0 270 618, EP 0 182448, EP 0 162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP 0 874057,EP 0 795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112, and EP 0 160457; Sanberg et al., XXth Int. Congress of the World Fed. Of Hemophilia(1992), and Lind et al., Eur. J. Biochem., 232:19 (1995).

In another embodiment, a method for treating hemophilia B in a humansubject, comprises administering an effective amount of areplication-defective virus composition of, in which the therapeuticproduct is Factor IX.

In another embodiment, a method for treating congestive heart failure ina human subject, comprises administering an effective amount of areplication-defective virus composition, in which the therapeuticproduct is insulin like growth factor or hepatocyte growth factor.

In another embodiment, a method for treating a central nervous systemdisorder in a human subject, comprises administering an effective amountof a replication-defective virus composition, in which the therapeuticproduct is nerve growth factor.

5.4. Monitoring Transgene Expression and Undesired Side Effects

5.4.1. Monitoring Transgene Expression

After administration of the replication-defective virus compositions ofthe invention, transgene expression can be monitored by any method knownto one skilled in the art. The expression of the administered transgenescan be readily detected, e.g., by quantifying the protein and/or RNAencoded by said transgene. Many methods standard in the art can be thusemployed, including, but not limited to, immunoassays to detect and/orvisualize protein expression (e.g., western blot, immunoprecipitationfollowed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), immunocytochemistry, immunohistochemical staining onsections etc.) and/or hybridization assays to detect gene expression bydetecting and/or visualizing respectively mRNA encoding a gene (e.g.,northern assays, dot blots, in situ hybridization, etc.). The viralgenome and RNA derived from the transgene can also be detected byQuantitative-PCR (Q-PCR). Such assays are routine and well known in theart. Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIP A buffer (1% NP-40 orTriton x-100, I% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1 to 4 hours) at 40° C., addingprotein A and/or protein G Sepharose beads to the cell lysate,incubating for about an hour or more at 40° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), incubating the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, incubating the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32p or 1251) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise.

ELISAs generally comprise preparing antigen, coating the well of a 96well microtiter plate with the antigen, adding the antibody of interestconjugated to a detectable agent such as an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable agent; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable agent may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art.

A phenotypic or physiological readout can also be used to assessexpression of a transgene. For example, the ability of a transgeneproduct to ameliorate the severity of a disease or a symptom associatedtherewith can be assessed. Moreover, a positron emission tomography(PET) scan and a neutralizing antibody assay can be performed. oreover,the activity a transgene product can be assessed utilizing techniqueswell-known to one of skill in the art. For example, the activity of atransgene product can be determined by detecting induction of a cellularsecond messenger (e.g., intracellular Ca2+, diacylglycerol, 1P3, etc.),detecting the phosphorylation of a protein, detecting the activation ofa transcription factor, or detecting a cellular response, for example,cellular differentiation, or cell proliferation or apoptosis via a cellbased assay. The alteration in levels of a cellular second messenger orphosphorylation of a protein can be determined by, e.g., immunoassayswell-known to one of skill in the art and described herein. Theactivation or inhibition of a transcription factor can be detected by,.e.g., electromobility shift assays, and a cellular response such ascellular proliferation can be detected by, e.g., trypan blue cellcounts, ³H-thymidine incorporation, and flow cytometry.

5.4.2. Monitoring Undesirable Side Effects/Toxicity

After administration of a replication-defective virus composition of theinvention to a patient, undesired side effects and/or toxicity can bemonitored by any method known to one skilled in the art fordetermination of whether to administer to the patient a pharmaceuticalcomposition comprising a dimerizer (described in Section 5.2.3) in orderto ablate or excise a transgene or to ablate the transcript of thetransgene, or inhibit its translation.

The invention provides for methods of determining when to administer apharmacological agent for ablating the therapeutic product to a subjectwho received a replication-defective virus composition encoding atherapeutic product and an ablator, comprising: (a) detecting expressionof the therapeutic product in a tissue sample obtained from the patient,and (b) detecting a side effect associated with the presence of thetherapeutic product in said subject, wherein detection of a side effectassociated with the presence of the therapeutic product in said subjectindicates a need to administer the pharmacological agent that inducesexpression of the ablator.

The invention also provides methods for determining when to administer apharmacological agent for ablating the therapeutic product to a subjectwho received a replication-defective virus composition encoding atherapeutic product and an ablator, comprising: detecting the level of abiochemical marker of toxicity associated with the presence of thetherapeutic product in a tissue sample obtained from said subject,wherein the level of said marker reflecting toxicity indicates a need toadminister the pharmacological agent that induces expression of theablator. Biochemical markers of toxicity are known in the art, andinclude clinical pathology serum measures such as, but not limited to,markers for abnormal kidney function (e.g., elevated blood urea nitrogen(BUN) and creatinine for renal toxicity); increased erythrocytesedimentation rate as a marker for generalized inflammation; low whiteblood count, platelets, or red blood cells as a marker for bone marrowtoxicity; etc. Liver function tests (lft) can be performed to detectabnormalities associated with liver toxicity. Examples of such lftsinclude tests for albumin, alanine transaminase, aspartate transaminase,alkaline phosphatase, bilirubin, and gamma glutamyl transpeptidase.

The invention further comprises methods for determining the presence ofDNA encoding the therapeutic gene product, its RNA transcript, or itsencoded protein in a tissue sample from the subject subsequent totreatment with the pharmacological agent that induces expression of theablator, wherein the presence of the DNA encoding the therapeutic geneproduct, its RNA transcript, or its encoded protein indicates a need fora repeat treatment with the pharmacological agent that inducesexpression of the ablator. One undesired side effect that can bemonitored in a patient that has received a replication-defective viruscomposition of the invention is an antibody response to a secretedtransgene product. Such an antibody response to a secreted transgeneproduct occurs when an antibody binds the secreted transgene product orto self antigens that share epitopes with the transgene product. Whenthe transgene product is an antibody, the response is referred to as an“anti-idiotype” response. When soluble antigens combine with antibodiesin the vascular compartment, they may form circulating immune complexesthat are trapped nonspecifically in the vascular beds of various organs,causing so-called immune complex diseases, such as serum sickness,vasculitis, nephritis systemic lupus erythematosus with vasculitis orglomerulonephritis.

In another, more generalized undesirable immune reaction to the secretedtransgene product, an antibody response to the transgene product resultsin a cross reacting immune response to one or more self antigens,causing almost any kind of autoimmunity. Autoimmunity is the failure ofan the immune system to recognize its own constituent parts as self,which allows an immune response against its own cells and tissues,giving rise to an autoimmune disease. Autoimmunity to the transgeneproduct of the invention can give rise to any autoimmune diseaseincluding, but not limited to, Ankylosing Spondylitis, Crohns Disease,Idiopathic inflammatory bowel disease, Dermatomyositis, Diabetesmellitus type-1, Goodpasture's syndrome, Graves' disease, Guillain-Barresyndrome (GBS), Anti-ganglioside, Hashimoto's disease, Idiopathicthrombocytopenic purpura, Lupus erythematosus, Mixed Connective TissueDisease, Myasthenia gravis, Narcolepsy, Pemphigus vulgaris, Perniciousanaemia, Psoriasis, Psoriatic Arthritis, Polymyositis, Primary biliarycirrhosis, Rheumatoid arthritis, Sjogren's syndrome, Temporal arteritis(also known as “giant cell arteritis”), Ulcerative Colitis (one of twotypes of idiopathic inflammatory bowel disease “IBD”), Vasculitis, andWegener's granulomatosis.

Immune complex disease and autoimmunity can be detected and/or monitoredin patients that have been treated with replication-defective viruscompositions of the invention by any method known in the art. Forexample, a method that can be performed to measure immune complexdisease and/or autoimmunity is an immune complex test, the purpose ofwhich is to demonstrate circulating immune complexes in the blood, toestimate the severity of immune complex disease and/or autoimmunedisease, and to monitor response after administration of the dimerizer.An immune complex test can be performed by any method known to one ofskill in the art. In particular, an immune complex test can be performedusing anyone or more of the methods described in U.S. Pat. No.4,141,965, U.S. Pat. No. 4,210,622, U.S. Pat. No. 4,210,622, U.S. Pat.No. 4,331,649, U.S. Pat. No. 4,544,640, U.S. Pat. No. 4,753,893, andU.S. Pat. No. 5,888,834, each of which is incorporated herein byreference in its entirety.

Detection of symptoms caused by or associated with anyone of thefollowing autoimmune diseases using methods known in the art is yetanother way of detecting autoimmunity or immune complex disease causedby a secreted transgene product that was encoded by areplication-defective virus composition administered to a human subject:Ankylosing Spondylitis, Crohns Disease, Idiopathic inflammatory boweldisease, Dermatomyositis, Diabetes mellitus type-I, Goodpasture'ssyndrome, Graves' disease, Guillain-Barre syndrome (GBS),Anti-ganglioside, Hashimoto's disease, Idiopathic thrombocytopenicpurpura, Lupus erythematosus, Mixed Connective Tissue Disease,Myasthenia gravis, Narcolepsy, Pemphigus vulgaris, Pernicious anaemia,Psoriasis, Psoriatic Arthritis, Polymyositis, Primary biliary cirrhosis,Rheumatoid arthritis, Sjogren's syndrome, Temporal arteritis (also knownas “giant cell arteritis”), Ulcerative Colitis (one of two types ofidiopathic inflammatory bowel disease “IBD”), Vasculitis, and Wegener'sgranulomatosis.

A common disease that arises out of autoimmunity and immune complexdisease is vasculitis, which is an inflammation of the blood vessels.Vasculitis causes changes in the walls of blood vessels, includingthickening, weakening, narrowing and scarring. Common tests andprocedures that can be used to diagnose vasculitis include, but are notlimited to blood tests, such as erythrocyte sedimentation rate,C-reactive protein test, complete blood cell count and anti-neutrophilcytoplasmic antibodies test; urine tests, which may show increasedamounts of protein; imaging tests such as X-ray, ultrasound,computerized tomography (CT) and magnetic resonance imaging (MRI) todetermine whether larger arteries, such as the aorta and its branches,are affected; X-rays of blood vessels (angiograms); and performing abiopsy of part of a blood vessel. General signs and symptoms ofvasculitis that can be observed in patients treated by the methods ofthe invention include, but are not limited to, fever, fatigue, weightloss, muscle and joint pain, loss of appetite, and nerve problems, suchas numbness or weakness.

When administration of a replication-defective virus composition of theinvention results in local transgene expression, localized toxicitiescan be detected and/or monitored for a determination of whether toadminister to the patient a pharmaceutical composition comprising adimerizer (described in Section 5.2.3) in order to ablate or excise atransgene or to ablate the transcript of the transgene, or inhibit itstranslation. For example, when administering to the retina areplication-defective virus composition that comprises a transgene unitencoding a VEGF inhibitor for treatment of age-related maculardegeneration, it is believed that VEGF may be neuroprotective in theretina, and inhibiting it could worsen eye-sight due to drop out ofganglion cells. Thus, after administration of such areplication-defective virus composition, eye-sight can be regularlymonitored and ganglion cell drop out can be detected by any method knownthe art, e.g., noninvasive imaging of retina. Moreover, VEGF inhibitionmay also depleted necessary micro vasculature in the retina, which canbe monitored using fluorescien angiography or any other method known inthe art.

In general, side effects that can be detected/monitored in a patientafter administration of a replication-defective virus of the inventionfor a determination of whether to administer a pharmaceuticalcomposition comprising a dimerizer (described in Section 5.2.3) to thepatient, include, but are not limited to bleeding of the intestine orany organ, deafness, loss of eye-sight, kidney failure, dementia,depression, diabetes, diarrhea, vomiting, erectile dysfunction, fever,glaucoma, hair loss, headache, hypertension, heart palpitations,insomnia, lactic acidosis, liver damage, melasma, thrombosis, priapismrhabdomyolysis, seizures, drowsiness, increase in appetite, decrease inappetite, dizziness, stroke, heart failure, or heart attack. Any methodcommonly used in the art for detecting the foregoing symptoms or anyother side effects can be employed.

Ablator Therapy; Once it has been determined that a transgene productthat was delivered to a patient by a method of the invention has causedundesirable side effects in a patient, a pharmaceutical compositioncomprising a dimerizer can be administered to a patient using any of theregimens, modes of administrations, or doses described in Section 5.2.3herein.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

6. EXAMPLE 1 Manufacturing of Recombinant AAV Vectors at Scale

A high yielding, recombinant AAV production process based uponpoly-ethylenimine (PEI)-mediated transfection of mammalian cells andiodixanol gradient centrifugation of concentrated culture supernatant.AAV vectors produced with the new process demonstrate equivalent orbetter transduction both in vitro and in vivo when compared to smallscale, cesium chloride (CsCl) gradient-purified vectors. In addition,the iodixanol gradient purification process described effectivelyseparates functional vector particles from empty capsids, a desirableproperty for reducing toxicity and unwanted immune responses duringpre-clinical studies.

Recently it was observed that, in contrast to AAV2, most other AAVserotypes are primarily released into the media of calciumphosphate-transfected production cultures and not retained in the celllysate (Vandenberghe, L. H., Lock, M., Xiao, R., Lin, J., Korn, M., andWilson, J. M. 2010. Heparin-dependent release of AAV into thesupernatant simplifies manufacturing, now published as “Efficientserotype-dependent Release of Functional Vector into the Culture MediumDuring Adeno-Associated Virus Manufacturing”, Hu Gene Ther, 21:1251-1257(October 2010)).

A scaled rAAV production method suitable for large animal studies, whichis based upon PEI transfection and supernatant harvestcan be utilizedThe method is high yielding, versatile for the production of vectorswith different serotypes and transgenes, and simple enough that it maybe performed in most laboratories with a minimum of specializedtechniques and equipment. This method is now published in Lock et al,Hum Gen Ther, 2010 October; 21 (10): 1259-71, which is incorporatedherein by reference.

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7. EXAMPLE 2 Cesium Purification of AAV Vectors

This example describes a new procedure for cesium chloride (CsCl)purification of AAV vectors from transfected cell pellets.

-   Day 1—Pellet Processing and CsCl Spin

1) Lysate preparation

-   -   Thaw cells from −80° C. freezer for 15 minutes at 37° C.    -   Resuspend the cell pellet in ˜20 mL of Resuspension Buffer 1(50        mM Tris, pH 8.0, 2 mM MgCl) for 40 plates of cells and for a        final volume of 20 mL, and place on ice.    -   Freeze/thaw 3 times (dry ice and ethanol bath/37° C. water        bath).    -   Add 100 μL of Benzonase (250 U/mL) per prep and invert gently,        incubate the samples at 37° C. for 20 minutes, inverting the        tube every 5 min    -   Add 6 mL of 5M NaCl to bring the final salt concentration to        1 M. Mix.    -   Spin at 8,000 rpm for 15 min at 4° C. in Sorval centrifuge.        Note: Ensure the Sorval is clean. After centrifugation,        sterilize tube with 70% before proceeding further. Transfer        supernatant to a new tube.    -   Spin again at 8,000 rpm for 15 min at 4° C. in Sorval. Note:        Ensure the Sorval is clean. After centrifugation, sterilize tube        with 70% before proceeding further.    -   Add 1.8 mL of 10% OGP for a final concentration of 0.5%, and mix        gently by inversion.

2) Cesium Chloride Step Gradient Purification

-   -   For each preparation, prepare two 2-tier gradients consisting of        7.5 mL of 1.5 g/mL CsCl and 15 mL 1.3 g/mL CsCl in Beckman SW-28        tubes (do not use ultraclear tubes). Load the less dense CsCl        first and then bottom load the heavier CsCl.    -   Add 15 mL of sample to the top of each gradient. Add sample        slowly to the side of the tube so as not to disturb the        gradient. Label the tubes with lot #.    -   Spin at 25,000 rpm at 15° C. for 20 hours minimum.

-   Day 2—Collect AAV band from 1st CsCl Spin and set up 2nd CsCI spin

1) Collect Band from CsCl Spin

-   -   Carefully remove the centrifuge tubes (A & B) out of the bucket,        taking care not to disturb the gradient. Secure the first        tube (A) on a tube holder.    -   Take a pre-sterilized 2 ft length of tygon-silicone tubing (1.6        mm inner diameter; Fisher NC9422080) fitted with two 1/16th inch        male luers (Fisher NC9507090) and insert 18G 1″ needles into the        luers.    -   Pierce the tube at a right angle as close to the bottom as        possible with one of the 18G 1″ needles (bevel facing up), and        clamp the tubing into the easy load rollers of the masterflex        pump. Gently increase the speed to˜1 mL/min Collect the first        4.5 mL into a 15 mL falcon tube and then start to collect        fractions (250 μL) into a 96 well plate (from tube A). Collect        48 fractions.    -   Run the rest of the gradient into a beaker containing a 20%        bleach solution and discard the needle/tubing assembly.    -   Take another pre-sterilized 2 ft length of tygon-silicone tubing        (1.6 mm inner diameter; Fisher NC9422080) fitted with two 1/16th        inch male luers (Fisher NC9507090) and insert 18G 1″ needles        into the luers for collecting fractions from second tube (from        tube B).    -   Repeat the entire harvest for the tube B. Discard the        needle/tubing assembly after use.

2) Read Refraction Index (RJ)

-   -   Using a multichannel pipetter, transfer 10 μL of each fraction        (of the 48 collected, first from 96-well plate A) to a fresh        plate (label with 1 to 48) and leave the remainder of the        fractions in the biosafety cabinet.    -   Take 5 μL of each fraction and read the RI using a        refractometer. The fractions containing AAV should have a        refractive index of 1.3740-1.3660. Read the RI down to 1.3650        and then pool the fractions in the biosafety cabinet with RI in        the 1.3740 to 1.3660 range. (Measure the total volume after        pooling both the 96-well plates belonging to tube 1 and 2. In        case there is still some space for adding more, add from wells        with RI of 1.375.)    -   Repeat this process for the second 96-well plate (from tube B).

3) Load the Second Gradient

-   -   The total pooled volume from each gradient (from tubes A and B)        should be 5-6 mL. Pool the two gradient harvests in a 50 mL        falcon tube and bring the volume to 13 mL with a 1.41 g/mL        solution of CsCl. Mix well with a pipette.    -   Using a 10 mL syringe and 18G needle, add the pooled first        gradient harvest to a 13 mL sealable centrifuge tube. The        solution should be added to the line on the neck of the tube        with no bubbles.    -   Seal the tube using the portable sealer, metal tube caps and        heat sink.    -   Squeeze the tube to test for leaks and then place in a Ti70.1        rotor with the appropriate balance. Insert the rotor caps and        lid and then spin at 60,000 rpm, 15° C. for 20 hours.

-   Day 3—Collect AAV band from 2nd CsCl Spin and Desalt

1) Collect Band from CsCl Spin

-   -   Carefully remove the centrifuge tube out of the bucket, taking        care not to disturb the gradient. Secure the tube on a tube        holder. At this point a single band should be visible after        bottom illumination about halfway up the tube.    -   Take a pre-sterilized 2 ft length of tygon-silicone tubing (1.6        mm inner diameter; Fisher NC9422080) fitted with two 1/16th inch        male luers (Fisher NC9507090) and insert 18G 1″ needles into the        luers. Use 1 length of tubing per prep.    -   Pierce the tube at a right angle as close to the bottom as        possible with one of the 18G 1″ needles (bevel facing up) and        clamp the tubing into the easy load rollers of the masterflex        pump. Pierce the tube again at the top with a second 18G needle.        Gently increase the speed to ˜1 mL/min and then start to collect        fractions (250 μL) into a 96 well plate. Collect the whole        gradient (-45 fractions).

2) Read Refractive Index (RI):

-   -   Using a multichannel pipetter, transfer 10 μL of each fraction        to a fresh plate and leave the remainder of the fractions in the        biosafety cabinet.    -   Take 5 μL of each fraction and read the RI using a        refractometer. The fractions containing AAV should have a        refractive index of 1.3750-1.3660. Read the RI down to 1.3650,        and then pool fractions with RI in range of 1.3750 to 1.3660.

3) Desalting: Amicon Ultra-I 5 centrifugal concentrators

-   -   In this procedure the vector is diluted with PBS and spun at low        speed through the 100 kDa MWCO filter device. Because of the        large molecular weight of AAV Particles (˜5000 kDa), the vector        is retained by the membrane and the salt passes through. Vector        can build up on the membranes, so rinsing is required at the        final stage.    -   Aliquot 50 mL PBS +35 mM NaCl into a 50 mL tube.    -   Dilute the pooled fractions from step 2 above with the PBS +35        mM NaCl to 15 mL total volume. Mix gently and add to Amicon        filter device.    -   Spin in a bench top Sorvall centrifuge at 2,000 to 4,000 rpm for        2 minutes. Because it is important to keep the level of the        liquid above the top of the filter surface (˜1.8 mL) at all        times so that the vector does not dry onto the membrane, it is        recommended that the lower speed spin is attempted first to        determine the flow rate of the sample. The goal is to reduce the        volume of the retentate to ˜1.8 mL. An additional short spines)        may be necessary to achieve this. If the volume does go below        that desired, bring it back to 1.8 mL with PBS+35 mM NaCl.    -   Add a further 13.2 mL PBS+35 mM NaCl, mix by pipette with the        retentate remaining in the device, and repeat the spinning        process described above. Continue this process until all the 50        mL PBS+35 mM NaCl aliquoted previously is spun through the        device.    -   Rinse the membrane with the final retentate (˜1.8 mL) by        repeatedly pipetting against the entire surface. Recover the        retentate into a suitably-sized sterile centrifuge tube using 1        mL and 200 μL Eppendorf tips (the 200 μL tip is for the final        retentate at the bottom of the device that is inaccessible to a        1 mL tip). Rinse the membrane twice using a minimum of 100 μL of        PBS+35 mM NaCl and pool it with your final retentate.    -   Determine the exact volume and add glycerol to 5%.

-   Aliquot into 5×25 μ: aliquots, 1×100 μL for archive, and the rest    into 105 μL aliquots.    -   Freeze immediately at −80 ° C.        Reagents Used in rAAV Purification    -   Resuspension buffer 1 [50 mM Tris (pH 8.0), 2 mM MgCl]: 50 mL 1        M Tris (pH 8.0), 2 mL/M MgCh to 948 mL MQ water, filter        sterilize.    -   1.5 g/mL CsCI solutions: dissolve 675 g of CsCI in 650 mL PBS        and adjust final volume to 1000 mL. Weigh 1 mL of the solution        to check the density. Filter sterilize the solution.    -   1.3 g/mL CsCI solutions: dissolve 405 g of CsCI in 906 mL PBS        and adjust final volume to 1000 mL. Weigh 1 mL of the solution        to check the density. Filter sterilize the solution.    -   10% (WN) Octyl-PD-glucopyranoside (OGP) (Sigma, 08001-10G):        Bring 10 grams to 100 mL with milliQ water. Filter sterilize the        solution.    -   Final formulation buffer: PBS+35 mM NaCl. To 1 liter sterile        PBS, add 7.05 mL sterile 5 M NaCl.    -   Sterile glycerol: Aliquot glycerol into 100 mL glass bottles.        Autoclave for 20 minutes on liquid cycle.

8. EXAMPLE 3 DNA Constructs for Preparation of PITA AAV Vectors

The invention is illustrated by Examples 3-5, which demonstrate thetight regulation of ablator expression using rapamycin, to dimerizetranscription factor domains that induce expression of Cre recombinase;and the successful inducible ablation of a transgene containing Crerecognition sites (loxP) in cells. The tight regulation of expression ofthe ablator is demonstrated in animal models.

The following are examples of DNA constructs DNA constructs and theiruse to generate replication-defective AAV vectors for use in accordancewith the PITA system of the invention is illustrated in the examplesbelow.

8.1. Constructs Encoding a Dimerizable Transcription Factor Domain Unitand an Ablation Unit

FIGS. 1A-B through FIGS. 5B are diagrams of the following DNA constructsthat can be used to generate AAV vectors that encode a dimerizabletranscription factor domain unit and an ablation unit: (1)pAAV.CMV.TF.FRB-I1RES-1xFKBP.Cre (FIGS. 1A-B); (2)pAAV.CMV.TF.FRB-T2A-2xFKBP.Cre (FIGS. 2A-B); (3)pAAV.CMVI73.TF.FRB-T2A-3xFKBP.Cre (FIGS. 3A-B); and (4)pAAV.CMV.TF.FRB-T2A-2xFKBP.ISce-I (FIGS. 4A-B).

A description of the various domains contained in the DNA constructsfollows:

-   ITR: inverted terminal repeats of AAV serotype 2 (168 bp). [SEQ ID    NO: 26]-   CMV: full cytomegalovirus (CMV) promoter; including enhancer. [SEQ    ID NO 27]-   CMV (173 bp): minimal CMV promoter, not including enhancer. [SEQ ID    NO: 28]-   FRB-TA fusion: fusion of dimerizer binding domain and an activation    domain of a transcription factor (900 bp, SEQ ID NO: 29). The    protein is provided herein as SEQ ID NO: 30. The FRB fragment    corresponds to amino acids 2021-2113 of FRAP (FKBP    rapamycin-associated protein, also known as mTOR [mammalian target    of rapamycin]), a phosphoinositide 3-kinase homolog that controls    cell growth and division. The FRAP sequence incorporates the single    point-mutation Thr2098Leu (FRAP_(L)) to allow use of certain    non-immunosuppressive rapamycin analogs (rapalogs). FRAP binds to    rapamycin (or its analogs) and FKBP and is fused to a portion of    human NF-KB p65 (190 amino acids) as transcription activator.-   ZFHD-FKBP fusion: fusion of a DNA binding domain and 1 copy of a    Dimerizer binding domain (1xFKBP; 732 bp), 2 copies of drug binding    domain (2xFKBP; 1059 bp), or 3 (3xFKBP;1389 bp) copies of drug    binding domain Immunophilin FKBP (FK506-binding protein) is an    abundant 12 kDa cytoplasmic protein that acts as the intracellular    receptor for the immunosuppressive drugs FK506 and rapamycin. ZFHD    is DNA binding domains composed of a zinc finger pair and a    homeodomain. Both fusion proteins contain N-terminal nuclear    localization sequence from human c-Myc at the 5′ end. See, SEQ ID    NO: 45.-   T2A: self cleavage peptide 2A (54 bp) (SEQ ID NO: 31).-   Z8I: 8 copies of the binding site for ZFHD (Z8) followed by minimal    promoter from the human interleukin-2 (1L-2) gene (SEQ ID NO: 32).    Variants of this promoter may be used, e.g., which contain from 1 to    about 20 copies of the binding site for ZFHD followed by a promoter,    e.g., the minimal promoter from IL-2.-   Cre: Cre recombinase. Cre is a type I topoisomerase isolated from    bacteriophage P1. Cre mediates site specific recombination in DNA    between two loxP sites leading to deletion or gene conversion (1029    bp, SEQ ID NO: 33).-   I-SceI: a member of intron endonuclease or homing endonuclease which    is a large class of meganuclease (708 bp, SEQ ID NO: 34). They are    encoded by mobile genetic elements such as introns found in bacteria    and plants. I-SceI is a yeast endonuclease involved in an intron    homing process. I-SecI recognizes a specific asymmetric 18 bp    element, a rare sequence in mammalian genome, and creates double    strand breaks. See, Jasin, M. (1996) Trends Genet., 12,224-228 .-   hGH poly A: minimal poly adenylation signal from human GH (SEQ ID    NO: 35).-   IRES: internal ribosome entry site sequence from ECMV    (encephalomyocarditis virus) (SEQ ID NO: 36).

8.2. Constructs Encoding Transgene Units

FIGS. 5A-B and FIGS. 6A-B are diagrams of the following DNA constructsfor generating an AAV vector encoding a transgene flanked by loxPrecognition sites for Cre recombinase:

(1) pENN.CMV.P1.loxP.Luc.SV40 (FIGS. 5A-B); and (2)pENN.CMV.P1.sce.Luc.SV40 (FIGS. 6A-B). A description of the variousdomains of the constructs follows:

ITR: inverted terminal repeats of AAV serotype 2 (SEQ ID NO: 26).

CMV: cytomegalovirus (CMV) promoter and enhancer regulating immediateearly genes expression (832 bp, SEQ ID NO: 27).

loxP: recognition sequences of Cre. It is a 34 bp element comprising oftwo 13 by inverted repeat flanking an 8 bp region which confersorientation (34 bp, SEQ ID NO: 37).

Ffluciferase: fire fly luciferase (1656 bp, SEQ ID NO: 38).

-   SV 40: late polyadenylation signal (239 bp, SEQ ID NO: 39).

I-SceI site: Sce1 recognition site (18 bp, SEQ ID NO: 25).

8.3. Constructs Encoding a Transgene Unit and a DimerizableTranscription Factor Domain Unit

FIG. 7 is a diagram of DNA construct for generating an AAV vector thatcontains a transgene unit and a dimerizable transcription factor domainunit. This plasmid provides, on AAV plasmid backbone containing anampicillin resistance gene, an AAV 5′ ITR, a transcription factor (TF)domain unit, a CMV promoter, an FRB (amino acids 2021-2113 of FRAP (FKBPrapamycin-associated protein, also known as mTOR [mammalian target ofrapamycin]), a phosphoinositide 3-kinase homolog that controls cellgrowth and division), a T2A self-cleavage domain, an FKBP domain, and ahuman growth hormone polyA site, a CMV promoter, a loxP site, aninterferon alpha coding sequence, and an SV40 polyA site. The ablationunit (cre expression cassette) can be located on a separate construct.This strategy could minimize any potential background level expressionof cre derived from upstream CMV promoter.

9. EXAMPLE 4 In Vitro Model for PITA

This example demonstrates that the DNA elements (units) engineered intothe AAV vectors successfully achieve tightly controlled inducibleablation of the transgene in cells. In particular, this example showsthat luciferase transgene expression can be ablated upon dimerizer(rapamycin) treatment of cells transfected with constructs containing atransgene unit (expressing luciferase and containing lox p sites), anablation unit (expressing Cre), and a dimerizable transcription factordomain unit.

Human embryonic kidney fibroblast 293 cells were seeded onto 12 wellplates. Transfection of the cells with various DNA constructs describedin section 9.1 herein was carried out the next day when the cell densityreached 90% confluency using lipofectamine 2000 purchased fromInvitrogen. A vector encoding enhanced green fluorescent protein (EGFP)was added at 10% of total DNA in each well to serve as internal controlfor transfection. The DNA suspended in DMEM was mixed with lipofectamine2000 to form DNA-lipid complex and added to 293 cells for transfectionfollowing instructions provided by Invitrogen Corporation. At 6 hourspost transfection, half of the wells were treated with rapamycin at afinal concentration of 50 nM. Culture medium (DMEM supplemented with 10%FBS) was replaced daily with fresh rapamycin. At 48 and 72 hour posttransfection, cells were washed once with PBS and then scraped out ofthe well, resuspended in lysis buffer supplied in Luciferase assay kitpurchased from Promega. The cell suspension was vortexed and the debrispun down. The luciferase activity was determined by mixing 10 μL of thelysate with 100 μL of the substrate and light emission per second readfrom a luminometer.

9.1. Constructs

The following constructs, most of which are described in Section 8,Example 1, were used to generate infectious, replication-defective AAVvectors:

-   1. pENN.AAV.CMV.RBG as a control, containing a CMV promoter and no    transgene-   2. pENN.CMV.P1.loxP.Luc.SV40 (FIGS. 5A-B)/pENN.AAV.CMV.RBG (CMV    promoter and no transgene)-   3. pENN.CMV.P1.loxP.Luc.SV40 (FIGS.    5A-B)/pAAV.TF.CMV.FRB-T2A-2xFKBP.Cre (FIGS. 2A-B)-   4. pENN.CMV.P1.loxP.Luc.SV40 (FIGS.    5A-B/pAAV.TF.CMV.FRB-IRES-FKBP.Cre (FIGS. 1A-B)-   5. pENN.CMV.P1.loxP.Luc.SV40 (FIGS.    5A-B)/pAAV.CMVI73.FRB-T2A-3xFKBP.Cre (FIGS. 3A-B)-   6. pENN.CMV.PI.loxP.Luc.SV40 (FIGS. 5A-B)/pENN.AAV.CMV.PI.Cre.RBG,    which expresses the Cre gene from a constitutive promoter

9.2. Results

The results at 48 hours are shown in FIG. 8A and the results at 72 hoursare shown in FIG. 8B. In the control (treatment 6), where Cre isconstitutively expressed, luciferase expression was ablatedindependently of rapamycin compared to the control expression ofluciferase without 10×P sites (treatment 2, cells transfected withluciferase construct). In contrast, in cells receiving the 10×P flankingluciferase construct plus one of the constructs carrying cre under thecontrol of PITA system (treatment 3,4 and 5), the level of the reportergene expression is comparable to the control in the absence ofdimerizer, rapamycin, indicating very little or no cre expression isinduced. However, upon induction by treatment with rapamycin, the levelof reporter gene expression in cells receiving PIT A controlled creconstructs were significantly reduced compared to the control (treatment2), indicating cre expression was activated. The results confirm thatthe expression of the ablator is specifically regulated by thedimerizer, rapamycin.

10. EXAMPLE 5 In Vivo Model for a Dimerizer-Inducible System

This example shows tight tissue-specific control of transgene expressionusing a liver-specific promoter that is regulated by thedimerizer-inducible system described herein. These data serves as amodel for tight regulation of the ablator in the PITA system.

Four groups of three mice received IV injection of AAV vectors encodingbicistronic reporter genes (GFP-Luciferase) at doses of 3×10¹⁰, 1×10¹¹and 3×10¹¹ particles of virus, respectively: Group 1 (G 1, G2, and G3)received AAV vectors expressing GFP Luciferase under the control ofubiquitous constitutive CMV promoter (see FIG. 9A for a diagram of theDNA construct). Group 2 (G4, G5, and G6) received co-injection of thefollowing 2 AAV vectors: (1) AAV vector expressing a dimerizabletranscription factor domain unit (FRB fused with p65 activation domainand DNA binding domain ZFHD fused with 3 copies of FKBP) driven by theCMV promoter (the DNA construct shown in FIG. 2B; and (2) AAV vectorexpressing GFP-Luciferase driven by a promoter induced by the dimerizedTF (see FIG. 12C for a diagram of the DNA constructs). Group 3 (G7, G8,and G9) received AAV vector expressing GFP-Luciferase under the controlof a liver constitutive promoter, TBG (see FIG. 9C for a diagram of theDNA construct). Group 4 (G10, G11, G12) received co-injection of thefollowing 2 AAV vectors: (1) AAV vector expressing a dimerizabletranscription factor domain unit (FRB fused with p65 activation domainand DNA binding domain ZFHD fused with 3 copies of FKBP) driven by theTBG promoter; and (2) AAV vector expressing GFP-Luciferase driven by apromoter induced by the dimerized TF (see FIG. 9D for a diagram of theDNA constructs).

About 2 weeks post virus administration, the mice were given IPinjection of the dimerizer, rapamycin, at the dose of 2 mg/kg. Startingthe next day the luciferase expression was monitored by Xenogen imaginganalysis. Approximately 24 hours post rapamycin injection, the mice wereIP injected with luciferin, the substrate for luciferase, thenanesthetized for imaging.

The mice that received 3×10¹¹ particles of virus had images taken 30 minpost luciferin injection (FIGS. 10A-D). For Group 1 mice that receivedvectors carrying GFP-Luciferase, expression driven by CMV promoter, theluciferase expression was observed in various tissues and predominantlyin lungs, liver and muscle (See FIG. 10A). In contrast, luciferaseexpression was restricted to liver in Group 3 mice, which receivedluciferase vector in which the expression was controlled by TBG promoter(see FIG. 10B). In Group 2 mice, the level of luciferase expression waselevated by more than 2 logs compared to level of pre-induction, and theexpression is predominantly in liver and muscle (see FIG. 10C). In Group4 mice, more than 100 fold of luciferase expression was induced andrestricted in the liver, compared to pre-inducement (see FIG. 10D).

The mice that received 1×10¹¹ particles of viruses, show results similarto that of high dose groups but with lower level of expression uponinduction, and predominantly in liver (see FIGS. 11A-D).

CONCLUSIONS

1. The dimerizer-inducible system is robust with peak level ofluciferase expression more than 2 logs over baseline and back to closeto baseline within a week (not shown).

2. Liver is the most efficient tissue to be infected when viruses weregiven IV.

3. Liver is also the most efficient tissue to be cotransduced with 2viruses which is critical for the dimerizer-inducible system to work.

4. The luciferase expression regulated by that dimerizer-induciblesystem with transcription factor expression controlled by CMV promoteris significantly higher in mouse liver than expression coming from CMVpromoter without regulation. This indicated that inducible promoter is astronger promoter in liver once it is activated compared to the CMVpromoter.

5. Luciferase expression was detected specifically in liver uponinduction by rapamycin in mice receiving vectors carrying the inducibleTBG promoter system. Luciferase expression mediated by theliver-specific regulatable vectors was completely dependent uponinduction by rapamycin and the peak level of luciferase expression iscomparable to that under the control of TBG promoter. This studyconfirmed that liver specific gene regulation can be achieved by AAVmediated gene delivery of liver specific dimerizer-inducible system.

11. EXAMPLE 6 PITA For Age-Related Macular Degeneration (AMD) Therapy

Intravitreal administration of a monoclonal antibody has proven to be aneffective therapy for AMD to slow down disease progression and improvevisual acuity in a subpopulation of patients. A key limitation of thisapproach, however, is the requirement for repeated intravitrealinjections. Gene therapy has the potential to provide long termcorrection and a single injection should be sufficient to achieve atherapeutic effect. FIGS. 12A-C show PITA DNA constructs for treatingAMD, containing transgene units comprising a VEGF antagonist, such as ananti-VEGF antibody (Avastin heavy chain (AvastinH) and Avastin lightchain (AvastinL); FIGS. 12B and 12C) or a soluble VEGF receptor (sFlt-1;FIG. 12A). Vectors comprising these DNA constructs can be delivered viasubretinal injection at the dose of 0.1-10 mg/kg. Ablation of transgeneexpression can be achieved by oral dimerizer administration if adverseeffects of long term anti-VEGF therapy are observed.

12. EXAMPLE 7 PITA for Liver Metabolic Disease Therapy

PITA is potentially useful for treating liver metabolic disease such ashepatitis C and hemophilia. FIG. 13A shows a PITA construct for treatinghemophilia A and/or B, containing a transgene unit comprising Factor IX.Factor VIII can also be delivered for treatment of hemophilia A and Brespectively (Factor VIII and IX for hemophilia A and B, respectively).The therapy could be ablated in patients if inhibitor formation occurs.FIG. 13B shows a PITA construct for delivery of shRNA targeting the IRESof HCV. A vector comprising this construct could be injected via amesenteric tributary of portal vein at the dose of 3×10¹² GC/kg. Theexpression of shRNA can be ablated if nonspecific toxicity of RNAinterference arises or the therapy is no longer needed.

13. EXAMPLE 8 PITA for Heart Disease Therapy

PITA could be utilized for heart disease applications including, but notlimited to, congestive heart failure (CHF) and myocardial infarction(MI). The treatment of CHF could involve the delivery of insulin likegrowth factor (IGF) or hepatocyte growth factor (HGF) using theconstructs shown in FIGS. 14A and 14B. For the treatment of myocardialinfarction, delivery of genes in the early stages of MI could protectthe heart from the deleterious effects of ischemia but allow ablation ofthe therapy when no longer required. Therapeutic genes for this approachinclude heme oxygenase-1 (HO-1) which can function to limit the extentof ischemic injury. Delivery methods for vector-mediated gene deliveryto the heart include transcutaneous, intravascular, intramuscular andcardiopulmonary bypass techniques. For the human, the optimalvector-mediated gene delivery protocol would likely utilize retrogradeor ante grade trans coronary delivery into the coronary artery oranterior cardiac vein.

14. EXAMPLE 9 PITA For Central Nervous System (CNS) Disease Therapy

Attractive candidates for the application of PITA in the central nervoussystem include neurotrophic factors for the treatment of Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),Huntington's disease and ocular diseases. FIG. 15 shows a PITA constructfor treating Alzheimer's disease, containing a transgene unit comprisingnerve growth factor (NGF). AAV vector-mediated gene delivery of NGF, iscurrently being studied in a Phase I clinical trial conducted byCeregene for the treatment of Alzheimer's disease. NGF is a neurotrophicfactor, which has been shown to be effective in reducing cholinergiccell loss in animal models of neurodegenerative disease and may beeffective in preventing loss of memory and cognitive abilities inpatients with AD. The delivery method for the approach consists ofbilateral, stereotactic injection to target the basal forebrain regionof the brain containing the nucleus basalis of Meynert (NBM). Due to thepotential for side-effects resulting in the need to end treatment,further engineering the construct to include PITA is warranted.

The application of PITA in the central nervous system for the treatmentof epilepsies could also be of value both due to the potential to ablategene expression once the issue surrounding the seizures becomes resolvedas well as due to the limited alternative approaches available for thetreatment of epilepsies that are unresponsive to drug therapy andsurgically difficult to treat. In these cases, in particular, deliverymethods involving sterotactic injection of vectors expressingtherapeutic genes, would be far less invasive than alternative surgicaltreatments. Candidates for gene expression could include galanin,neuropeptide Y (NPY) and glial cell line-derived neurotrophic factor,GDNF, which have been shown to have therapeutic effects in animal modelsof epilepsy. Other applications include to deliver nerve growth factor(NGF) for Alzheimer's and aromatic L-amino acid decarboxylase (ADCC) forParkinson's Disease.

15. EXAMPLE 10 PITA for HIV Therapy

Naturally induced neutralizing antibody against HIV has been identifiedin the sera of long term infected patients. As an alternative to activevaccine approaches, which have resulted in inefficient induction butsufficient levels of neutralizing antibody delivered by AAV, PITA is apromising approach to deliver anti-HIV neutralizing antibody for passiveimmunity therapy. See FIG. 16. The construct design is similar toavastin gene delivery for AMD therapy (see FIGS. 12B and 12C). A vectorcomprising a construct encoding an antibody regulated by the liverspecific promoter (TBG) could be injected into the liver at a dose of3×10¹² GC/kg. Alternatively, a vector comprising a construct carrying aubiquitous CB7 promoter driving antibody expression could be deliveredby intramuscular injection at a dose of 5×10¹² GC/mL for up to 20injections into the quadriceps or biceps muscle. The therapy can beablated if it is no longer needed or if toxicity develops due toinduction of anti-drug antibody.

16. EXAMPLE 11

The DNA constructs described in the following example may be used toprepare replication-defective AAV viruses and virus compositionsaccording to the invention.

Open reading frames encoding for various endonucleases were codonoptimized and de novo synthesized by GeneArt. Ablator expression andtarget plasmids were produced using standard molecular biologicalcloning techniques. Transfections were performed in HEK293 cells usingLipofectamine™ 2000 transfection reagent (Life Technologies). Alltransfections were performed using optimal transfection conditions asdefined in transfection reagent protocol. Briefly, 200-250 ng plasmidDNA (excluding transfection control plasmid) was complexed withlipofectamine and added to cells in 96 well plates. DNA quantities wereconsistent across all conditions by supplementation with an unrelatedplasmid containing the same promoter as test plasmids. Transfectioncomplexes were incubated with cells for 4-6 hours as transfectionreagent protocol before the addition of FBS supplemented media.Transfected cells were incubated at 37° C. for 24-72 hours. Followingincubation, cells were assayed for reporter gene expression usingPromega Dual Luciferase detection kit according to the manufacturer'sinstructions on a BioTek Clarity platereader and renilla luciferase wasused to control for transfection efficiency. All samples were performedin quadruplicate and standard errors of the mean were calculated.

A. Coexpression of Wild-Type FokI Ablates Expression of Transgene moreEffectively than Delivery of FokI Protein

The amino acid sequence of the FokI enzyme is provided in SEQ ID NO: 12,wherein amino acids 1 to 387 are the DNA binding domain and amino acids387 to 584 are the catalytic domain. The codon optimized FokI sequenceis provided in SEQ ID NO:1.

FIG. 18 illustrates that wild-type FokI effective ablated expression ofthe luciferase reporter gene following contrasfection into HEK295 cells(FIG. 18A bar 2), while only partial ablation was observed when FokIprotein was delivered to the cells (FIG. 18A, bar 3).

In a dose-dependent experiment, the FokI expression vector contained theFokI catalytic domain fused to a zinc finger DNA binding domain (ZFHD).This construct, which is 963 bp, is provided in SEQ ID NO: 21 and iscomposed of base pairs 1 to 366 bp ZFHD, 367 to 372 bp linker, and 373to 963 bp FokI catalytic domain. The resulting expression productcomprises amino acids 1 to 122 (ZFHD), amino acids 123-124 are a linkerand amino acids 125 to 321 are from the FokI catalytic domain. FIG. 18Billustrates that increasing the concentration of FokI resulted in dosedependent ablation of Luc reporter. No ablation sites were required tobe engineered into the transcription unit containing the transgene inthis illustration, as luciferase contains multiple native FokI sites.This provides support for the use of the PITA system using a transfectedFokI enzyme directed to specific ablation sites in a transcription unitcontaining a transgene for delivery to the cell.

B. Chimeric Engineered FokI Tethered to Non-Cognate Recognition Site onthe DNA by the Zinc Finger—Homeodomain Effectively Ablates Expression ofLuc Reporter Gene

The plasmid contructs in this example contains either the FokI catalyticdomain (198 amino acids (SEQ ID NO: 14), corresponding to amino acids387 to 584 of the full-length protein) (untethered FokI) or a ZFHD-FokIcatalytic domain of 963 bp as described in Part A above (tethered FokI).Even at the highest concentration, the catalytic domain of FokI which isun-tethered to DNA does have no effect on expression of Luc reportergene (FIG. 19A). Chimeric engineered FokI tethered to DNA via fusionwith ZFHD effectively ablated expression of luciferase reporter in adose dependent manner when increasing concentrations of ZF-HD-FokIexpression plasmid were cotransfected into HEK293 cells (FIG. 19B).

This supports the use of the PITA system and the additional safetyelement provided by a chimeric enzyme directed to specific ablationsites in a transcription unit containing a transgene for delivery to thecell.

C. DNA Binding Specificity of Chimeric FokI can be Reproducibly Changedby Fusion with Various Classes of Heterologous DNA Binding Domains andAblation of Target Transgene can be Further Improved by Addition ofHeterologous NLS

This example illustrates that the zinc finger homeodomain (ZFHD) is notthe only domain suitable for altering the specificity of ablationmediated by a chimeric engineered enzyme. FokI effectively ablatedexpression of luciferase reporter in a dose dependent manner when HTHDNA binding domain was fused to FokI catalytic domain (FIG. 20A). In aseparate experiment (FIG. 27B), the activity of HTH-FokI was furtherimproved by adding heterologous NLS at the N-terminus of the HTH-FokIcoding sequence.

The HTH-FokI Catalytic domain (SEQ ID NO:5), is composed of 1-171 bp HTHfrom Gin (a serine recombinase), a linker (bp 172-177), and a FokIcatalytic domain (178-768 bp) derived from codon-optimized FokI. Theresulting chimeric enzyme (SEQ ID NO: 6) contains aa 1-57 of HTH fromGin, a linker (aa 58-59), and a FokI catalytic domain (amino acids60-256).

FIGS. 20A-20B are bar charts illustrating that the DNA bindingspecificity of chimeric FokI can be reproducible changed by fusion withanother classes of heterologous DNA binding domains and ablation oftarget transgene can be further improved by the additional of aheterologous nuclear localiazation signal (NLS). FIG. 20A illustratesthe results of co-transfection of pCMV.Luciferase with increasingconcentrations of an expression plasmid encoding FokI tethered to DNAvia an HTH fusion (6.25, 12.5, 25, 50, and 100 ng). The first bar is acontrol showing 50 ng pCMV.Luciferase alone. FIG. 20B pCMV.Luciferasewith increasing concentrations of an expression plasmid encoding anHTH-FokI fusion, which further has a NLS at its N-terminus.

17. EXAMPLE 12

Although not illustrated here, other chimeric enzymes have been madeusing the techniques described herein:

An AAV plasmid containing SV40 T-Ag NLS-Helix-turn-helix (HTH) from Gin(192 bp, SEQ ID NO:7), which includes the nuclear localization signal(1-24 bp) of SV40 T-Ag and HTH from Gin, a serine recombinase (25-192bp). In the resulting enzyme (SEQ ID NO:8), amino acids 1-8 are from theSV40 T-Ag NLS and amino acids 9-64 are the HTH from Gin;

An AAV plasmid containing SV40 T-Ag NLS-HTH-FokI Catalytic domain (789bp, SEQ ID NO:9), which includes the SV40 T-Ag NLS (bp 1-24), the HTHfrom Gin (bp 25-192), a linker (bp 193-198), and the catalytic domain ofthe FokI (bp 199-789). In the resulting chimeric enzyme (SEQ ID NO:10),amino acids 1-8 are from the SV40 T-Ag NLS, amino acids 9-64 are HTHfrom G in, amino acids 65-66 are linker residues, and amino acids 67-263are the FokI catalytic domain.

An AAV plasmid containing a SV40 T-Ag NLS-ZFHD-FokI catalytic domain(984 bp) was prepared (SEQ ID NO: 23), which includes the SV40 T-Ag NLS(bp 1-24), the zinc finger homodomain (bp 25-387), a linker (bp388-393), and the FokI catalytic domain (bp acids 394-984). In theresulting chimeric enzyme (SEQ ID NO: 21, 328 aa), amino acids 1-8 arethe SV40 T-Ag NLS, amino acids 9-129 are the ZFHD, amino acids 130-131are linker residues, and amino acids 132-138 are FokI catalytic domain.

These and other constructs can be used to prepare viruses according themethod of the invention for use in a virus composition and the PITAsystem.

18. EXAMPLE 13 Use Of Replication-Defective AAV Virus Compostion inTreatment of HIV

This composition could be potentially used as a safety mechanism in thetreatment of HIV. Recently, broadly neutralizing antibodies fromlong-term non-progressors, individuals which maintain an HIV⁺ status forseveral decades without progression to AIDS, have been identified byseveral research groups.

All coding regions of the neutralizing antibody to HIV (HIV NAb) areplaced between the inverted terminal repeats (ITRs) of the AAV. If theoverall size of the constructs are below 4.7 kb (including the twoITRs), they are packaged into the AAV capsid. The AAV serotype capsidchosen will depend of the level of gene expression, the method ofdelivery and the extent of biodistribution from the injection siterequired. In addition, the constitutive promoters used for expression ofthe HIV NAb (and potentially the parts of the inducible system in theone small molecule situation) would depend on the tissue type targeted.In the following example of a potential clinical study the vectorserotype chosen would be AAV8 administered by intravenous injectionwhich would enable utilization of the liver specific promoter TBG.

In HIV⁺ patients, administration of AAV vectors expressing one or moreof these HIV neutralizing antibodies would lead to long-term, high levelexpression of one or more broadly HIV NAb and would reduce viral loadand potentially prevent acquisition of HIV. In this situation,individuals would receive intravenous injection of two AAV vectors at adose of 5×10¹² genome copies/kilogram of each vector. Contained withinthe two AAV vectors would be the HIV neutralizing antibody under controlof a constitutive promoter, allowing expression to occur rapidlyfollowing administration of the vector.

A. Heterodimer and Two Small Molecules

Following the first signs of potential toxicity to the HIV NAb, thefirst small molecule drug would be administered to induce expression ofthe components of the inducible system, in this case the DNA bindingdomain linked to FKBP and FRAP_(L) linked to the catalytic domain of aendonuclease enzyme. This would allow the system to be primed for actionshould further toxicity to the HIV NAb develop. If toxicity levelscontinue to rise then initiation of endonuclease activity would beinduced by administration of a second small molecule drug which wouldlead to the formation of an active enzyme and ablation of HIV NAb geneexpression.

B. Heterodimer and One Small Molecule

Also under the control of constitutive expression would be the elementsof the rapamycin inducible system, FKBP and FRAP_(L). Followingadministration of the AAV vectors, patients would be closely monitoredat regular intervals for several years. If toxicity to the HIV NAbdevelops then delivery of rapamycin or a rapalog would be implemented.IV administration of 1 mg/kg rapamycin/rapalog in the first instancewith the potential to increase to repeated dosing would be administeredto ablate expression of the HIV antibody. Toxicity and HIV antibodylevels would be closely monitored until expression of the

HIV NAb had reached undetectable levels. Therefore, the ablation of geneexpression of the HIV NAb would provide a safety switch to ablate geneexpression should insurmountable toxicity occur.

In one aspect, the invention provides a replication-defective viruscomposition suitable for use in human subjects in which the viral genomehas been engineered to contain: (a) a first transcription unit thatencodes a therapeutic product in operative association with a promoterthat controls transcription, said unit containing at least one ablationrecognition site; and (b) a second transcription unit that encodes anablator specific for the at least one ablation recognition site inoperative association with a promoter, wherein transcription and/orablation activity is controlled by a pharmacological agent, e.g., adimerizer. For example, one suitable pharmacologic agent may berapamycin or a rapamycin analog. The virus composition may contain twoor more different virus stocks.

In one aspect, the invention provides a replication-defective viruscomposition suitable for use in human subjects in which the viral genomecomprises (a) a first transcription unit that encodes a therapeuticproduct in operative association with a promoter that controlstranscription, said first transcription unit containing an ablationrecognition site; and a second transcription unit that encodes anablator specific for the ablation recognition site in operativeassociation with a promoter, wherein transcription and/or ablationactivity is controlled by a pharmacological agent. The firsttranscription unit can contains more than one ablation recognition site.Where the genome comprises more than one ablation recognition site, saidmore than one ablation recognition site comprising a first ablationrecognition site and a second ablation recognition site which differsfrom said first ablation recognition site, said virus further comprisinga first ablator specific for the first ablation recognition site and asecond ablator specific for the second recognition site.

In one embodiment, the transcription, bioactivity and/or the DNA bindingspecificity of the ablator is controlled by a regulatable system. Theregulatable system can be selected from a tet-on/off system, a tetR-KRABsystem, a mifepristone (RU486) regulatable system, a tamoxifen-dependentregulatable system, a rapamycin-regulatable system, or an ecdysone-basedregulatable system.

In one embodiment, the ablator is selected from the group consisting of:an endonuclease, a recombinase, a meganuclease, or a zinc fingerendonuclease that binds to the ablation recognition site in the firsttranscription unit and excises or ablates DNA and an interfering RNA, aribozyme, or an antisense that ablates the RNA transcript of the firsttranscription unit, or suppresses translation of the RNA transcript ofthe first transcription unit. In one specific embodiment, the ablator isCre and the ablation recognition site is loxP, or the ablator is FLP andthe ablation recognition site is FRT.

In an embodiment, the ablator is a chimeric engineered endonuclease,wherein the virus composition comprises (i) a first sequence comprisingthe DNA binding domain of the endonuclease fused to a binding domain fora first pharmacological agent; and wherein the virus composition furthercomprises (ii) a second sequence encoding the nuclease cleavage domainof the endonuclease fused to a binding domain for the firstpharmacological agent, wherein the first sequences (i) and the secondsequence (ii) are each in operative association with at least onepromoter which controls expression thereof The chimeric engineeredendonuclease can be contained within a single bicistronic open readingframe in the second transcription unit, said transcription unit furthercomprising a linker between (i) and (ii). Optionally, the sequence (ii)has an inducible promoter. In another embodiment, the fusionpartners/fragments of the chimeric engineered endonuclease are containedwithin separate open reading frames. In one embodiment, each of thefirst sequence and the second sequence are under the control of aconstitutive promoter and the ablator is bioactivated by the firstpharmacological agent.

The coding sequence for the ablator may further comprise a nuclearlocalization signal located 5′ or 3′ to the ablator coding sequence.

In one embodiment, the DNA binding domain is selected from the groupconsisting of a zinc finger, helix-turn-helix, a HMG-Box, Stat proteins,B3, helix-loop-helix, winged helix-turn-helix, leucine zipper, a wingedhelix, POU domains,and a homeodomain.

In still another embodiment, the endonuclease is selected from the groupconsisting of a type II restriction endonuclease, an intronendonuclease, and serine or tyrosine recombinases. In one specificembodiment, the ablator is a chimeric FokI enzyme.

In yet another embodiment, in a replication-defective virus compositionof the invention, the viral genome further comprises a third and afourth transcription unit, each encoding a dimerizable domain of atranscription factor that regulates an inducible promoter for theablator, in which: (c) the third transcription unit encodes the DNAbinding domain of the transcription factor fused to a binding domain forthe pharmacological agent in operative association with a firstpromoter; and (d) the fourth transcription unit encodes the activationdomain of the transcription factor fused to a binding domain for thepharmacological agent in operative association with a second promoter.The first promoter of (c) and the second promoter of (d) areindependently selected from a constitutive promoter and an induciblepromoter. In another embodiment, the first and second promoters are bothconstitutive promoters and the pharmacological agent is a dimerizer thatdimerizes the domains of the transcription factor. In still a furtherembodiment, one of the first promoter and the second promoters is aninducible promoter. The the third and fourth transcription units can bea bicistronic unit containing an IRES or furin-2A.

In one embodiment, the pharmacological agent is rapamycin or a rapalog.In one embodiment, the virus is an AAV. Such an AAV may be selected fromamong, e.g., AAV1, AAV6, AAV7, AAV8, AAV9 and rh10. Still other virusesmay be used to generate the DNA constructs and replication-defectiveviruses of the invention including, e.g., adenovirus, herpes simplexviruses, and the like.

In one embodiment, the therapeutic product is an antibody or antibodyfragment that neutralizes HIV infectivity, soluble vascular endothelialgrowth factor receptor-1 (sFlt-1), Factor VIII, Factor IX, insulin likegrowth factor (IGF), hepatocyte growth factor (HGF), heme oxygenase-1(HO-1), or nerve growth factor (NGF).

In one embodiment of the replication-defective virus composition, thefirst transcription unit and the second transcription unit are ondifferent viral stocks in the composition. Optionally, the firsttranscription unit and the second transcription unit are in a firstviral stock and the a second viral stock comprises a second ablator(s).

In one embodiment, a recombinant DNA construct comprises a first andsecond transcription unit flanked by packaging signals of a viralgenome, in which: (a) a first transcription unit that encodes atherapeutic product in operative association with a promoter thatcontrols transcription, said first transcription unit containing atleast one ablation recognition site; and (b) a second transcription unitthat encodes an ablator specific for the at least one ablationrecognition site in operative association with a promoter that inducestranscription in response to a pharmacological agent. The packagingsignals flanking the transcription units may be an AAV 5′ invertedterminal repeats (ITR) and a AAV 3′ ITR. Optionally, the AAV ITRs areAAV2, or AAV1, AAV6, AAV7, AAV8, AAV9 or rh10 ITRs. In one embodiment,the first transcription unit is flanked by AAV ITRs, and the second,third and fourth transcription units are flanked by AAV ITRs.Optionally, the transcription units are contained in two or more DNAconstructs.

In one embodiment, the therapeutic product is an antibody or antibodyfragment that neutralizes HIV infectivity, soluble vascular endothelialgrowth factor receptor-1 (sFlt-1), Factor VIII, Factor IX, insulin likegrowth factor (IGF), hepatocyte growth factor (HGF), heme oxygenase-1(HO-1), or nerve growth factor (NGF).

In one embodiment, the promoter that controls transcription of thetherapeutic product is a constitutive promoter, a tissue-specificpromoter, a cell-specific promoter, an inducible promoter, or a promoterresponsive to physiologic cues.

A method is described for treating age-related macular degeneration in ahuman subject, comprising administering an effective amount of thereplication-defective virus composition as described herein, in whichthe therapeutic product is a VEGF antagonist.

A method is provided for treating hemophilia A in a human subject,comprising administering an effective amount of thereplication-defective virus composition as described herein, in whichthe therapeutic product is Factor VIII.

A method is provided for treating hemophilia B in a human subject,comprising administering an effective amount of thereplication-defective virus composition as described herein, in whichthe therapeutic product is Factor IX.

A method is provided for treating congestive heart failure in a humansubject, comprising administering an effective amount of thereplication-defective virus composition as described herein, in whichthe therapeutic product is insulin like growth factor or hepatocytegrowth factor.

A method is provided for treating a central nervous system disorder in ahuman subject, comprising administering an effective amount of thereplication-defective virus composition as described herein, in whichthe therapeutic product is nerve growth factor.

A method is provided for treating HIV infection in a human subject,comprising administering an effective amount of thereplication-defective virus composition as described herein in which thetherapeutic product is a neutralizing antibody against HIV.

A replication-defective virus is provided herein for use in controllingdelivery of the transgene product. The product may be selected from thegroup consisting of a VEGF antagonist, Factor IX, Factor VIII, insulinlike growth factor, hepatocyte growth factor, nerve growth factor, and aneutralizing antibody against HIV.

A genetically engineered cell is provided which comprises areplication-defective virus or a DNA construct as provided herein. Thegenetically engineered cell may be selected from a plant, bacterial ornon-human mammalian cell.

A method is provided for determining when to administer apharmacological agent for ablating a therapeutic product to a subjectwho received the replication-defective virus as provided hereincontaining a therapeutic product and an ablator, comprising: (a)detecting expression of the therapeutic product in a tissue sampleobtained from the patient, and (b) detecting a side effect associatedwith the presence of the therapeutic product in said subject, whereindetection of a side effect associated with the presence of thetherapeutic product in said subject indicates a need to administer thepharmacological agent that induces expression of the ablator.

A method is provided for determining when to administer apharmacological agent for ablating a therapeutic product to a subjectwho received the replication-defective virus composition as describedherein encoding a therapeutic product and an ablator, comprising:detecting the level of a biochemical marker of toxicity associated withthe presence of the therapeutic product in a tissue sample obtained fromsaid subject, wherein the level of said marker reflecting toxicityindicates a need to administer the pharmacological agent that inducesexpression of the ablator.

These methods may further comprise determining the presence of DNAencoding the therapeutic gene product, its RNA transcript, or itsencoded protein in a tissue sample from the subject subsequent totreatment with the pharmacological agent that induces expression of theablator, wherein the presence of the DNA encoding the therapeutic geneproduct, its RNA transcript, or its encoded protein indicates a need fora repeat treatment with the pharmacological agent that inducesexpression of the ablator.

The invention further provides a replication-defective virus asdescribed herein for use in controlling delivery of the transgeneproduct.

In another embodiment, the invention provides a genetically engineeredcell, comprising a replication-defective virus or a DNA construct asdescribed herein. Such a cell may be a plant, yeast, fungal, insect,bacterial, non-human mammalian cells, or a human cell.

In yet a further embodiment, the invention provides a method ofdetermining when to administer a pharmacological agent for ablating atherapeutic product to a subject who received the replication-defectivevirus as described herein encoding a therapeutic product and an ablator,comprising: (a) detecting expression of the therapeutic product in atissue sample obtained from the patient, and (b) detecting a side effectassociated with the presence of the therapeutic product in said subject,wherein detection of a side effect associated with the presence of thetherapeutic product in said subject indicates a need to administer thepharmacological agent that induces expression of the ablator. In still afurther embodiment, the invention provides a method of determining whento administer a pharmacological agent for ablating a therapeutic productto a subject who received the replication-defective virus composition asdescribed herein encoding a therapeutic product and an ablator,comprising: detecting the level of a biochemical marker of toxicityassociated with the presence of the therapeutic product in a tissuesample obtained from said subject, wherein the level of said markerreflecting toxicity indicates a need to administer the pharmacologicalagent that induces expression of the ablator.

EXAMPLE 14 Generation of 10xZF Constructs

Open reading frames encoding for various endonucleases were codonoptimized and de novo synthesized by GeneArt (Invitrogen). Ablatorexpression and target reporter plasmids were produced using standardmolecular biological cloning techniques. Transfections were performed inHEK293 cells using Lipofectamine™ 2000 transfection reagent (LifeTechnologies). All transfections were performed using optimaltransfection conditions as defined in transfection reagent protocol.Briefly, 150-200 ng plasmid DNA (excluding transfection control plasmid)was complexed with lipofectamine and added to cells in 96 well plates.DNA quantities were consistent across all conditions by supplementationwith an unrelated plasmid containing the same promoter as test plasmids.Transfection complexes were incubated with cells for 4 hours astransfection reagent protocol before the addition of FBS supplementedmedia. Transfected cells were incubated at 37° C. for 24-48 hours.Following incubation, cells were assayed for reporter gene expressionusing Promega Dual Luciferase detection kit according to themanufacturer's instructions on a BioTek Clarity plate reader and renillaluciferase was used to control for transfection efficiency. All sampleswere performed in quadruplicate and standard errors of the mean werecalculated.

A. Generation of 10xZF Expression Plasmid

-   1. Once random sequence 1 was generated and determined to be    targetable by standard zinc finger design methodologies, the design    of the nucleases was performed using the resources available from    The Zinc Finger (ZF) Consortium and the zinc finger database (these    are publically available. See, e.g.,    bindr.gdcb.iastate.edu:8080/ZiFDB/). Based on the breakdown of the    28-32 bp sequence into 10× 3 bp ZF binding sites, ZF domains which    bind to each of the 3 bp sequences were identified.

The following 28-32 bp sequences were generated:

[SEQ ID NO: 806] GGTCGATGTTCGCAACGTCGATCGTACGTGCA. For this sequence,there are two reading frames which are targetable by different zincfingers: GGT-CGA-TGT-TCG-CAA-CGT-CGA-TCG-TAC-GTG-CA-targetableG-GTC-GAT-GTT-CGC-AAC-GTC-GAT-CGT-ACG-TGC-A-targetable (the experimentsdescribed herein were generated for this sequence)

[SEQ ID NO: 817]: GGTCGGCGACGCGTAATCGTCGATTGGCGTAC. For this sequence,there are two reading frames which are targetable by different zincfingers. G-GTC-GGC-GAC-GCG-TAA-TCG-TCG-ATT-GGC-GTA-C-targetableGG-TCG-GCG-ACG-CGT-AAT-CGT-CGA-TTG-GCG-TAC-targetable

[SEQ ID NO: 801] GGTCGGCGACGCGAATCGTCGATTGGCGTAC. For this sequence,there are two reading frames which are targetable by different zincfingers. G-GTC-GGC-GAC-GCG-TAA-TCG-TCG-ATT-GGC-GTA-C-targetableGG-TCG-GCG-ACG-CGT-AAT-CGT-CGA-TTG-GCG-TAC-targetable

[SEQ ID NO: 802] GGTCGGCGACGCGTATCGATTGGCGTAC. For this sequence, thereis one potential targetable by a zinc finger:GGT-CGG-CGA-CGC-GTA-TCG-ATT-GGC-GTA-C-targetable

[SEQ ID NO: 803] ACTATTCGCACGCCGTACGATAGTCGGCGCGA. For this sequence,there are two reading frames targetable by zinc fingers:ACT-ATT-CGC-ACG-CCG-TAC-GAT-AGT-CGG-CGC-GA-targetable andA-CTA-TTC-GCA-CGC-CGT-ACG-ATA-GTC-GGC-GCG-A-targetable.

For any 3 bp sequence for which only one known ZF sequence was availablewithin the consortium, this sequence was chosen for the final protein.This was the case for 3 out of the 10xZF domains.

Provided within the consortium database was a table of previously testedcombination of 2 or 3 ZFs linked together in a particular orientationfor binding to DNA. Within our random sequence 1, there are two regionswhich contain previously tested combinations. One is a 3× 3 bp stretchand the other is a 2× 3 bp stretch. Where a previous combination of ZFswas reported to work, this was incorporated into the 10xZF sequence.

For 3 out of 10xZF sequences, only the recognition helix of the DNAbinding region was included in the consortium. For 7 out of the 10xZFsequences, the full ZF sequence was available in the consortium.Therefore, the full sequence of the ZFs where only the recognition helixwas available through the consortium had to be generated. In addition,to prevent high level sequence homology between the 10xZF protein, theconserved protein sequence of the ZF protein domain was varied betweenone of the two conserved sequences in the consortium:

P start conserved sequence [SEQ ID NO: 745] PGEKPYKCPECGKSFS-------HQRTHT start conserved sequence [SEQ ID NO: 807] TGEKPFQCRICMRNFS-------HLRTHThe ZFs were linked N terminus to C terminus directly as there innateprotein sequence would allow for the correct structure of the finalprotein.

The 10xZF sequence was then linked to the catalytic domain of the FokIenzyme (the DNA binding domain was removed from the wild-type sequence)through a standard linker sequence on the C terminal of the 10xZFprotein.

In the working examples below, an ablator encoded by the sequence:10xZF-FokI_Cat nucleotide sequence: SEQ ID NO: 59, is illustrated:

ATGGGCGAGAAGCCCTACAAGTGCCCTGAGTGCGGCAAGAGCTTCAGCCAGAGAAGAAGCCTGGGCCACCACCAGCGTACGCACCCCGGCGAGAAACCTTATAAGTGTCCCGAATGTGGCAAGTCCTTCAGCAAGAAGAACGACCTGACCCGGCACCAGCGGACACACCCCGGGGAAAAGCCATACAAATGTCCAGAGTGTGGGAAGTCTTTCTCCAGCCGGCGGACCTGCAGAGCCCATCAGAGAACACATACCGGGGAGAAGCCTTTCCAGTGCCGGATCTGCATGAGAAACTTCAGCGTGCGGCACAACCTGACCAGACACCTGAGGACCCATACCGGCGAAAAACCCTTTCAGTGCAGAATCTGTATGCGGAACTTCTCCGACCGGACCAGCCTGGCCCGGCATCTGAGAACTCATCCTGGGGAAAAGCCCTATAAGTGTCCAGAATGCGGGAAATCCTTTAGCGACAGCGGCAACCTGCGGGTGCACCAGAGGACTCATCCAGGCGAGAAACCCTACAAATGCCCCGAATGCGGAAAGTCATTCTCCCACACCGGCCATCTGCTCGAGCATCAGCGGACCCACACTGGGGAGAAACCATTTCAGTGTCGCATCTGTATGAGGAATTTCAGCACCAACCAGGCCCTGGGCGTGCACCTGAGAACACACCCAGGCGAGAAGCCTTACAAGTGTCCAGAGTGCGGAAAGTCATTTTCCGTGCGCCACAATCTGACACGGCATCAGCGCACCCATCCCGGCGAGAAGCCTTACAAATGCCCCGAGTGTGGCAAATCTTTCAGTGACCGGACCTCTCTGGCCAGACATCAGAGGACACACGGCACTAGTGGCAAGCAGCTGGTGAAAAGCGAGCTGGAAGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAATACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACCCCACCCAGGACAGAATCCTGGAAATGAAGGTCATGGAATTTTTCATGAAGGTGTACGGCTACCGGGGCGAGCACCTGGGCGGCAGCAGAAAACCCGACGGCGCCATCTACACCGTGGGCAGCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACAGCGGCGGCTACAACCTGCCCATCGGACAGGCCGACGAGATGCAGAGATACGTGGAAGAGAACCAGACCCGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCCGGCTGAACCACATCACCAACTGCAACGGCGCTGTGCTGAGCGTGGAAGAACTGCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAAGAAGTGCGGCGGAAGTTCAACAACGGCGAGATCAACTTCTGATAG.

In this construct, bpi-84: zinc finger N1 (recognition helix QRRSLGH, Pform, binds to TGC); bp 85-168: zinc finger N2 (recognition helixKKNDLTR, P form, binds to ACG); bp 169-252: zinc finger N3 (recognitionhelix SRRTCRA, P form, binds to CGT); bp 253-336: zinc finger N4(recognition helix VRHNLTR, T form, binds to GAT); bp 337-420: zincfinger N5 (recognition helix DRTSLAR, T form, binds to GTC), by 421-504bp: zinc finger N6 (recognition helix DSGNLRV, P form, binds to AAC); by505-588: zinc finger N7 (recognition helix HTGHLLEM, P form, binds toCGC); bp 589-672: zinc finger N8 (recognition helix TNQALGV, T form,binds to GTT); bp 673-756: zinc finger N9 (recognition helix VRHNLTR, Pform, binds to GAT); bp 757-840: zinc finger N10 (recognition helixDRTSLAR, P form, binds to GTC); bp 841-855: 5 amino acid linker; bp856-1443: FokI catalytic domain.

The transcribed illustrative ablator has the sequence: 10xZF-FokI_Catamino acid sequence: SEQ ID NO: 60:

MGEKPYKCPECGKSFSQRRSLGHHQRTHPGEKPYKCPECGKSFSKKNDLTRHQRTHPGEKPYKCPECGKSFSSRRTCRAHQRTHTGEKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDRTSLARHLRTHPGEKPYKCPECGKSFSDSGNLRVHQRTHPGEKPYKCPECGKSFSHTGHLLEHQRTHTGEKPFQCRICMRNFSTNQALGVHLRTHPGEKPYKCPECGKSFSVRHNLTRHQRTHPGEKPYKCPECGKSFSDRTSLARHQRTHGTSGKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF.1-28 aa-zinc finger N1 (recognition helix QRRSLGH, P form, binds toTGC); 29-56 aa-zinc finger N2 (recognition helix KKNDLTR, P form, bindsto ACG); 57-84 aa-zinc finger N3 (recognition helix SRRTCRA, P form,binds to CGT); 85-112 aa-zinc finger N4 (recognition helix VRHNLTR, Tform, binds to GAT); 113-140 aa-zinc finger N5 (recognition helixDRTSLAR, T form, binds to GTC); 141-168 aa-zinc finger N6 (recognitionhelix DSGNLRV, P form, binds to AAC); 169-196 aa-zinc finger N7(recognition helix HTGHLLEM, P form, binds to CGC); 197-224 aa-zincfinger N8 (recognition helix TNQALGV, T form, binds to GTT); 225-252aa-zinc finger N9 (recognition helix VRHNLTR, P form, binds to GAT);253-280 aa-zinc finger NiO (recognition helix DRTSLAR, P form, binds toGTC); 281-284 aa-5 amino acid linker; and 285-481 aa-FokI catalyticdomain.

In another embodiment, the 10xZF sequence is linked to the catalyticdomain of the FokI enzyme (the DNA binding domain was removed from thewild-type sequence) through a linker sequence on the N terminus of the10xZF protein.

N-linked FokI_Cat-10xZF nucleotide sequence: SEQ ID NO: 808:

ATGAAGCAGCTGGTGAAAAGCGAGCTGGAAGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAATACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACCCCACCCAGGACAGAATCCTGGAAATGAAGGTCATGGAATTTTTCATGAAGGTGTACGGCTACCGGGGCGAGCACCTGGGCGGCAGCAGAAAACCCGACGGCGCCATCTACACCGTGGGCAGCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACAGCGGCGGCTACAACCTGCCCATCGGACAGGCCGACGAGATGCAGAGATACGTGGAAGAGAACCAGACCCGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCCGGCTGAACCACATCACCAACTGCAACGGCGCTGTGCTGAGCGTGGAAGAACTGCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAAGAAGTGCGGCGGAAGTTCAACAACGGCGAGATCAACTTCGGCACTAGTGGCGGCGAGAAGCCCTACAAGTGCCCTGAGTGCGGCAAGAGCTTCAGCCAGAGAAGAAGCCTGGGCCACCACCAGCGTACGCACCCCGGCGAGAAACCTTATAAGTGTCCCGAATGTGGCAAGTCCTTCAGCAAGAAGAACGACCTGACCCGGCACCAGCGGACACACCCCGGGGAAAAGCCATACAAATGTCCAGAGTGTGGGAAGTCTTTCTCCAGCCGGCGGACCTGCAGAGCCCATCAGAGAACACATACCGGGGAGAAGCCTTTCCAGTGCCGGATCTGCATGAGAAACTTCAGCGTGCGGCACAACCTGACCAGACACCTGAGGACCCATACCGGCGAAAAACCCTTTCAGTGCAGAATCTGTATGCGGAACTTCTCCGACCGGACCAGCCTGGCCCGGCATCTGAGAACTCATCCTGGGGAAAAGCCCTATAAGTGTCCAGAATGCGGGAAATCCTTTAGCGACAGCGGCAACCTGCGGGTGCACCAGAGGACTCATCCAGGCGAGAAACCCTACAAATGCCCCGAATGCGGAAAGTCATTCTCCCACACCGGCCATCTGCTCGAGCATCAGCGGACCCACACTGGGGAGAAACCATTTCAGTGTCGCATCTGTATGAGGAATTTCAGCACCAACCAGGCCCTGGGCGTGCACCTGAGAACACACCCAGGCGAGAAGCCTTACAAGTGTCCAGAGTGCGGAAAGTCATTTTCCGTGCGCCACAATCTGACACGGCATCAGCGCACCCATCCCGGCGAGAAGCCTTACAAATGCCCCGAGTGTGGCAAATCTTTCAGTGACCGGACCTCTCTGGCCAGACATCAGAGGACACAC.

This nucleic acid sequence encodes, at: FokI catalytic domain (1-594bp), 4 amino acid linker (595-606 bp), zinc finger N1 (recognition helixQRRSLGH, P form, binds to TGC) (607-687 bp), zinc finger N2 (recognitionhelix KKNDLTR, P form, binds to ACG) (688-771 bp), zinc finger N3(recognition helix SRRTCRA, P form, binds to CGT) (772-855 bp), zincfinger N4 (recognition helix VRHNLTR, T form, binds to GAT) (856-939bp), zinc finger N5 (recognition helix DRTSLAR, T form, binds to GTC)(940-1023 bp), zinc finger N6 (recognition helix DSGNLRV, P form, bindsto AAC) (1024-1107 bp), zinc finger N7 (recognition helix HTGHLLEM, Pform, binds to CGC) (1108-1191 bp), zinc finger N8 (recognition helixTNQALGV, T form, binds to GTT) (1192-1275 bp), zinc finger N9(recognition helix VRHNLTR, P form, binds to GAT) (1276-1359 bp), zincfinger N10 (recognition helix DRTSLAR, P form, binds to GTC) (1360-1443bp).

The transcribed N-linked 10xZF-FokI_Cat amino acid sequence: SEQ ID NO:809: is as follows.

MKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFGTSGGEKPYKCPECGKSFSQRRSLGHHQRTHPGEKPYKCPECGKSFSKKNDLTRHQRTHPGEKPYKCPECGKSFSSRRTCRAHQRTHTGEKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDRTSLARHLRTHPGEKPYKCPECGKSFSDSGNLRVHQRTHPGEKPYKCPECGKSFSHTGHLLEHQRTHTGEKPFQCRICMRNFSTNQALGVHLRTHPGEKPYKCPECGKSFSVRHNLTRHQRTHPGEKPYKCPECGKSFSDRTSLARHQRTH.

This chimeric endonuclease is: FokI catalytic domain (1-198 aa), 5 aminoacid linker (199-202 aa), zinc finger N1 (recognition helix QRRSLGH, Pform, binds to TGC) (203-229 aa), zinc finger N2 (recognition helixKKNDLTR, P form, binds to ACG) (230-257 aa), zinc finger N3 (recognitionhelix SRRTCRA, P form, binds to CGT) (258-285 aa), zinc finger N4(recognition helix VRHNLTR, T form, binds to GAT) (286-313 aa), zincfinger N5 (recognition helix DRTSLAR, T form, binds to GTC) (314-341aa), zinc finger N6 (recognition helix DSGNLRV, P form, binds to AAC)(342-369 aa), zinc finger N7 (recognition helix HTGHLLEM, P form, bindsto CGC) (370-397 aa), zinc finger N8 (recognition helix TNQALGV, T form,binds to GTT) (398-425 aa), zinc finger N9 (recognition helix VRHNLTR, Pform, binds to GAT) (426-453 aa), and zinc finger N10 (recognition helixDRTSLAR, P form, binds to GTC) (454-481 aa).

B. Generation of Reporter Plasmids for 10x ZF-FokI Cat Protein

In order to study the efficiency of the 10x ZF expression plasmid, aseries of luciferase reporter plasmids were also designed with theunique 32 bp sequence: [SEQ ID NO: 806] GGTCGATGTTCGCAACGTCGATCGTACGTGCAgenerated as described in A above. These vectors include:

pITA-030, which contains, from 5′ to 3′: a cytomegalovirus (CMV)immediate early (IE) enhancer and promoter (bp 1-832), a Promega™ intron(833-1029 bp), the 32 bp sequence in sense orientation (1069-1100 bp), aspacer (1101-1106 bp), the 32 bp sequence in inverted orientation(1107-1138 bp), a Kozak sequence (1147-1152 bp), the coding sequence fora luciferase reporter gene (1153-2802 bp), and an SV40 polyA sequence(2822-3061 bp), of SEQ ID NO: 811.

pITA-031, which contains, from 5′ to 3′: a CMV promoter IEenhancer/promoter (1-832 bp), a Promega™ intron (833-1029 bp), the 32 bpsequence in direct/sense orientation (1069-1100 bp), a spacer (1101-1106bp), the 32 bp sequence in inverted orientation (1107-1138 bp), a Kozaksequence (1147-1152 bp), the coding sequence for a luciferase reportergene (1153-2802 bp), a 32 bp sequence in sense orientation (2815-2846bp), a spacer (2847-2852 bp), the 32 bp sequence in inverted orientation(2853-2884 bp), and an SV40 polyA sequence (2893-3131 bp), of SEQ ID NO:812.

pITA-032, which contains, from 5′ to 3′: a CMV IE enhancer/promoter(1-832 bp), a Promega™ intron (833-1029 bp), the 32 bp sequence in senseorientation (1069-1100 bp), a Kozak sequence (1115-1120 bp), the codingsequence for a luciferase reporter gene (1121-2770 bp), and an SV40polyA sequence (2791-3029 bp), of SEQ ID NO: 813.

pITA-033, which contains, from 5′ to 3′: a CMV IE enhancer/promoter(1-832 bp), a Promega™ intron (833-1029 bp), the 32 bp sequence in senseorientation (1069-1100 bp), a Kozak sequence (1115-1120 bp), the codingsequence for a luciferase reporter gene (1121-2770 bp), the 32 bpsequence in sense orientation (2783-2814 bp), and an SV40 polyA sequence(2829-3067 bp), of SEQ ID NO: 814.

pITA-034, which contains, from 5′ to 3′: a CMV IE enhancer/promoter(1-832 bp), a Promega™ intron (833-1029 bp), the 32 bp sequence in senseorientation (1069-1100 bp), a Kozak sequence (1115-1120 bp), the codingsequence for a luciferase reporter gene (1121-2770 bp), the 32 bpsequence in direct orientation (2789-2820 bp), and an SV40 polyAsequence (2829-3067 bp), of SEQ ID NO: 815.

pITA-005 (control) contains from 5′ to 3′: a CMV IE enhancer/promoter(1-832 bp), a Promega™ intron (833-1029 bp), a Kozak sequence (1077-1082bp), the coding sequence for a luciferase reporter gene (1083-2732 bp),and an SV40 polyA sequence (2752-2991 bp).

These reporter plasmids were studied in dose-dependent studies, of SEQID NO: 810.

(A) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected into 293 cells with 50 ng pCMV.32 bp-Luciferase(pITA-032, single specific site for 10xZF protein at 5′ end ofluciferase gene). The cells were assayed for reporter gene expression asdescribed above 48 hours post-transfection.

(B) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected into 293 cells with 50 ng pCMV.32bp-Luciferase-32 bp (pITA-033, single specific site for 10xZF protein at5′ and at 3′ end of luciferase gene in head-to-tail orientation). Thecells were assayed for reporter gene expression as described above 48hours post-transfection.

(C) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected into 293 cells with 50 ng pCMV.32bp-Luciferase-32 bp (pITA-034, single specific site for 10xZF protein at5′ end of luciferase and a single specific inverted site at the 3′ endof luciferase gene). The cells were assayed for reporter gene expressionas described above 48 hours post-transfection.

(D) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected with 50 ng pCMV.32 bpSpacer32 bp-Luciferase(pITA-030, single combined specific site for 10xZF containing a 32 bpsite with a spacer separating an identical but inverted 32 bp at the 5′end of the luciferase gene). The cells were assayed for reporter geneexpression as described above 48 hours post-transfection.

(E) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected with 50 ng pCMV.32 bpSpacer32bp-Luciferase-32bpSpacer32 bp (pITA-031, single combined specific site for 10xZFcontaining a 32 bp site with a spacer separating an identical butinverted 32 bp at both the 5′ end and the 3′ end of the luciferasegene). The cells were assayed for reporter gene expression as describedabove 48 hours post-transfection. (F) Increasing concentrations of theexpression plasmid encoding FokI tethered to DNA via 10xZF fusion (1.56,3.13, 6.25, 12.5, 25, 50 and 100 ng) were co-transfected with 50 ngpCMV. Luciferase (pITA-005, luciferase expression plasmid containing nospecific site for 10xZF). The cells were assayed for reporter geneexpression as described above 24 hours post-transfection.

(G) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion (1.56, 3.13, 6.25, 12.5, 25, 50 and 100ng) were co-transfected with 50 ng pCMV. Luciferase (pITA-005,luciferase expression plasmid containing no specific site for 10xZF).The cells were assayed for reporter gene expression as described above48 hours post-transfection.

In each of (A)-(E), dose-dependent ablation was observed for all fivereporter plasmids, demonstrating that the 10x ZF design provided by thisinvention requires only one 32 bp sequence to be present within thereporter and, therefore, within the vector in the applications describedherein.

Different orientations of the reporter plasmid were studied indose-dependent studies.

(H) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion with the 10xZF domain at the N-terminusof the protein (1.56, 3.13, 6.25, 12.5, 25 and 50 ng) wereco-transfected into 293 cells with 50 ng pCMV.32 bpSpacer32 bpLuciferase(pITA-030, single combined specific site for 10xZF containing a 32 bpsite with a spacer separating an identical but inverted 32 bp at the 5′end of the luciferase gene). The cells were assayed for reporter geneexpression as described above 48 hours post-transfection.

(I) Increasing concentrations of the expression plasmid encoding FokItethered to DNA via 10xZF fusion with the FokI catalytic domain at theN-terminus of the protein (1.56, 3.13, 6.25, 12.5, 25 and 50 ng) wereco-transfected into 293 cells with 50 ng pCMV.32 bpSpacer32 bpLuciferase(pITA-030, single combined specific site for 10xZF containing a 32 bpsite with a spacer separating an identical but inverted 32 bp at the 5′end of the luciferase gene). The cells were assayed for reporter geneexpression as described above 48 hours post-transfection.

All publications, patents, and patent applications cited in thisapplication, as well as priority applications PCT/US2011/030213, filedMar. 28, 2011 and U.S. Patent Application No. 61/318,755 and theSequence Listing, are hereby incorporated by reference in theirentireties as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A composition for AAV-mediated delivery of atherapeutic product having a controlled transgene expression ablationsystem, said composition comprising (a) an AAV vector containing anucleic acid molecule comprising: (i) a nucleic acid sequence encoding atherapeutic product operably linked to a promoter that controlstranscription; and (ii) at least one endonuclease ablation site whichcomprises a sequence of at least 30 nucleic acid base pairs which arespecifically recognized by at least ten (10X) zinc fingers, said atleast one endonuclease ablation site being located at least 5′ to thesequence encoding the therapeutic product; (b) at least one ablatorwhich comprises a chimeric endonuclease comprising at least ten copiesof a zinc finger domain linked to a functional endonuclease catalyticdomain in operative association with a promoter, wherein transcriptionand/or ablation activity is induced in response to a pharmacologicalagent, said at least ten (10×) zinc finger domain specificallyrecognizing said at least about 30 base pair sequence in said at leastone endonuclease ablation site and comprising at least 10 independentlyselected recognition helices.
 2. The composition according to claim 1,wherein the endonuclease catalytic domain is a FokI catalytic domain. 3.The composition according to claim 1, wherein the at least 10independently selected recognition helices are all different.
 4. Thecomposition according to claim 3, wherein the at least 10 independentlyselected recognition helices include different recognition helices tothe same triplet of base pairs in the ablation site.
 5. The compositionaccording to claim 1, wherein the at least 10 independently selectedrecognition helices contain 1 to 3 helices which are the same.
 6. Thecomposition according to claim 1, wherein the at least 30 base pairsequence in the endonuclease ablation site comprise at least 32 basepairs.
 7. The composition according to claim 1, wherein the moleculecomprises a first endonuclease ablation site and a second endonucleaseablation site, wherein said first and said second endonuclease ablationsite may be the same or different unique sequence.
 9. The compositionaccording to claim 7, wherein the first endonuclease ablation site andthe second endonuclease ablation site are both located 5′ to the codingsequence and are separated by a spacer sequence.
 10. The compositionaccording to claim 9, wherein the second endonuclease ablation site isspacer sequence is inverted.
 11. The composition according to claim 9,wherein the spacer sequence is a non-coding sequence.
 12. Thecomposition according to claim 7, wherein said second endonucleaseablation site is located 3′ to the coding sequence.
 13. The compositionaccording to claim 1, wherein the catalytic domain of the endonucleaseis linked to the zinc finger catalytic domain sequence on the N or Cterminus of the zinc finger catalytic domain sequence
 14. Thecomposition according to claim 13, wherein the catalytic domain islinked to the zinc finger catalytic domain sequence via a linkersequence.
 15. The composition according to claim 1, wherein thecatalytic domain of the endonuclease is located within the zinc fingerdomain sequence.
 16. The composition according to claim 1, wherein thenucleic acid molecule consists of a double-stranded DNA molecule,wherein the at least one endonuclease ablation site is on a first strandof the DNA molecule and at least a second endonuclease ablation islocated on the second strand of the DNA molecule, wherein said secondendonuclease ablation site is distinct from said endonuclease ablationsaid on the first strand and is specifically and selectively recognizedby a different zinc fingers.
 17. The composition according to claim 1,wherein the at least 30 base pair sequence is selected from the groupconsisting of:   (i) SEQ ID NO: 806:5′-GGTCGATGTTCGCAACGTCGATCGTACGTGCA-3′;  (ii) SEQ ID NO: 801:5′-GGTCGGCGACGCGAATCGTCGATTGGCGTAC-3′   and (iii) SEQ ID NO: 803:5′-ACTATTCGCACGCCGTACGATAGTCGGCGCGA-3′.


18. The composition according to claim 1, wherein said zinc fingerdomain comprises a nucleic acid sequence encoding at least ten zincfingers consisting of : (a) a first N-terminal zinc finger comprising arecognition helix which specifically binds to TGC; (b) second zincfinger comprising a recognition helix which specifically binds to ACG;(c) a third zinc finger comprising a recognition helix whichspecifically binds to CGT; (d) a fourth zinc finger comprising arecognition helix which specifically binds to GAT; (e) a fifth zincfinger comprising a recognition helix which specifically binds to GTC;(f) a sixth zinc finger comprising a recognition helix whichspecifically binds to AAC; (g) a seventh zinc finger comprising arecognition helix which binds to CGC; (h) an eighth zinc fingercomprising a recognition helix that specifically binds to GTT; (i) anninth zinc finger comprising a recognition helix that specifically bindsto GAT; and (j) a tenth zinc finger comprising a recognition helix whichspecifically binds to GTC.
 19. The composition according to claim 18,wherein: the recognition helix of (a) which specifically binds to TGC isselected from the group consisting of: ARNTLVH, QRRSLGH, QARSLRA,QQRSLKN, and QNRSLAH, QGRSLRA, RARNLTL, RGRNLEM, RKRNLIM, RMRNLII,RNRNLVL, RRRNLHL, RRRNLTL, RSRNLDI, RSRNLLL, and RSRNLTL (SEQ ID NO:658-673); the recognition helix of (b) which specifically binds to ACGis selected form the group consisting of: KNNDLTR; KRIDLQR; RKHDLNM;RRQTLRQ; KGNDLTR; PSQTLAWQ; RNITLVR, RSHDLTV, ASADLTR, QNATRKR, QSGDLTR,RSQTLAQ; and RTDTLRD (SEQ ID NO: 104-119); the recognition helix of (c)which specifically binds to CGT is selected from the group consisting ofRSQTRKT (SEQ ID NO: 154) and SRRTCRA (SEQ ID NO: 155); the recognitionhelix of (d) which specifically binds to GAT is selected from the groupconsisting of VRHNLTR, ISHNLAR, ISSNLQR, LGNNLKR, LNSNLAR, LSTNLTR,LTHNLRR, QSSNLAR, RSDALIQ, SKQALAV, TGQQLRV, TKQRLVV, TRQRLRI, TSANLSR,TSGNLVR, TSQMLVV, TSSNLSR, TTSNLRR, VGHNLSR, VGSNLTR (SEQ ID NO:251-270); the recognition helix of (e) which specifically binds to GTCis selected from the group consisting of DRTSLAR, DHSSLKR, APSSLRR,DATQLVR, DPGALVR, DPTSLNR, DRSALAR, DRSALSR, DRSSLRR, DRTPLNR, DRTPLQN,EGGALRR, ESGALRR, NTSLLRR, RSDVLSE, TGAVLRR, TGAVLTR, TKKILTV, TKSLLAR,TMAVLRR, TRAVLRR, TSTILAR, TSTLLKR, and TSTLLNR (SEQ ID NO: 530-553);the recognition helix of (f) which specifically binds to AAC is selectedfrom the group consisting of DRSNRKT, DSGNLRV, GASALRQ, GASALRS,GGTALRM, GGTALVM, GHTALAL, GHTALRH, GHTALRN, GPTALVN, and HRTNLIA (SEQID NO: 63-73); the recognition helix of (g) which specifically binds toCGC is HTGHLLE (SEQ ID NO: 151); the recognition helix of (h) whichspecifically binds to GTT is selected from the group consisting ofHKSSLTR, TNQALGV, AATALRR, HHNSLTR, HSSSLVR, IKAILTR, INHSLRR, IRTSLKR,MNSVLKR, MTSSLRR, QATLLRR, QSSALTR, THTVLAR, TKPVLKI, TNSVLGR, TRHSLGR,TSGALTR, TSGSLTR, TSGSLVR, TSTLLKR, TSTRLDI, TTALLKR, TTSALTR, TTTVLAR,and VGGSLNR (SEQ ID NO: 583-607); the recognition helix of (i) whichspecifically binds to GAT is selected from the group consisting ofISHNLAR, VRHNLTR, ISSNLQR, LGNNLKR, LNSNLAR, LSTNLTR, LTHNLRR, QSSNLAR,RSDALIQ, SKQALAV, TGQQLRV, TKQRLVV, TRQRLRI, TSANLSR, TSGNLVR, TSQMLVV,TSSNLSR, TTSNLRR, VGHNLSR, and VGSNLTR (SEQ ID NO: 251-270); and therecognition helix of (j) which specifically binds to GTC is selectedfrom the group consisting of DRTSLAR, DHSSLRKR, APSSLRR, DATQLVR,DPGALVR, DPTSLNR, DRSALAR, DRSALSR, DRSSLRR, DRTPLNR, DRTPLQN, EGGALRR,ESGALRR, NTSLLRR, RSDVLSE, TGAVLRR, TGAVLTR, TKKILTV, TKSLLAR, TMAVLRR,TRAVLRR, TSTILAR, TSTLLKR, and TSTLLNR (SEQ ID NO: 530-553).
 20. Thecomposition according to claim 19, wherein each of the zinc fingers(a)-(j) has the selected recognition domain inserted in a zinc fingerconstruct selected from: SEQ ID NO: 745:(N-terminus)-PGEKPYKCPECGKSFS-XXXXXXX-HQRTH (carboxy terminus), COOH andSEQ ID NO: 807: (N-terminus)-TGEKPFQCRICMRNFS-XXXXXXX-HLRTH(carboxy terminus), COOH,

wherein XXXXXXX is the zinc finger recognition domain.
 21. Thecomposition according to claim 19, wherein: the recognition helix of (a)which specifically binds to TGC is QRRSLGH (SEQ ID NO: 663); therecognition helix of (b) which specifically binds to ACG is KKNDLTR (aa29-56 of SEQ ID NO: 60); the recognition helix of (c) which specificallybinds to CGT is SRRTCRA (SEQ ID NO: 155); the recognition helix of (d)which specifically binds to GAT is VRHNLTR (SEQ ID NO: 270); therecognition helix of (e) which specifically binds to GTC is DRTSLAR (SEQID NO: 64); the recognition helix of (f) which specifically binds to AACis DSGNLRV (SEQ ID NO: 540); the recognition helix of (g) whichspecifically binds to CGC is HTGHLLE (SEQ ID NO: 151); the recognitionhelix of (h) which specifically binds to GTT is TNQALGV (aa 197-224 ofSEQ ID NO: 60); the recognition helix of (j) which specifically binds toGAT is VRHNLTR (SEQ ID NO: 270); and the recognition helix of (k) whichspecifically binds to GTC is DRTSLAR (SEQ ID NO: 540).
 22. Thecomposition according to claim 1, wherein the zinc finger sequence islinked to the sequence encoding a FokI catalytic domain through asequence encoding five amino acids GTSGK (SEQ ID NO: 805), whereby theresulting chimeric ablator cuts 6 bp directly following the zinc fingerbinding site.
 23. The composition according to claim 1, wherein thetranscription of the promoter is controlled by a rapamycin-regulatablesystem.
 24. The composition according to claim 20, wherein which thepharmacological agent is rapamycin or a rapalog.
 25. The compositionaccording to claim 1, wherein the ablator (b) is controlled by acassette that is activated by a transcription factor following beingdimerized by a pharmacologic agent, said cassette comprising twotranscription units, wherein: (c) one of said two transcription unitsencoding the DNA binding domain of the transcription factor fused to abinding domain for the pharmacological agent in operative associationwith a first promoter; and (d) a second of said two transcription unitsencoding the activation domain of the transcription factor fused to abinding domain for the pharmacological agent in operative associationwith a second promoter.
 26. The composition of claim 25, wherein thefirst promoter of (c) and the second promoter of (d) are independentlyselected from a constitutive promoter and an inducible promoter.
 27. Thecomposition of claim 26, wherein the first and second promoters are bothconstitutive promoters and the pharmacological agent is a dimerizer thatdimerizes the domains of the transcription factor.
 28. The compositionof claim 19, wherein one of the first promoter and the second promotersis an inducible promoter.
 29. A method for delivering an ablatabletransgene to a subject, said method comprising administering thecomposition according to claim 1 to the subject.